Coating process for microfluidic sample arrays

ABSTRACT

A differentially coated device for conducting a plurality of nano-volume specified reactions, the device comprising a platen having at least one exterior surface modified to a specified physicochemical property, a plurality of nano-volume channels, each nanovolume channel having at least one interior surface in communication with the at least one exterior surface. Methods for preparing and using such devices are also provided, as well as a method of registering a location of a dispenser array in relation to a microfluidic array. Quantities related to a vector displacement from the alignment position to a fixed position on the one of the dispenser array and the microfluidic array is determined. The quantities thus determined are used to guide positioning of the dispenser array relative to the microfluidic array.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/300,100 filed Jun. 9, 2014, which is a continuation of U.S.application Ser. No. 11/198,882, filed Aug. 4, 2005, and claims priorityto U.S. application No. 60/608,231, filed Sep. 9, 2004, and claimspriority to U.S. application No. 60/599,217, filed Aug. 4, 2004, whichdisclosures are herein incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to techniques for transferring smallvolumes of liquid, and more specifically to calibration of dispensersrelative to nanoliter sample volumes in an array. The present inventionalso relates to processes and the devices for spatially selectivechemical modification or coating of the surfaces of a substrate orthrough-hole array plate, such as may be used in microfluidic ornanofluidic storage and analysis systems or other applications.

BACKGROUND ART

Various systems are known for performing a large number of chemical andbiological storage assays and synthesis operations. One approach uses anassay chip having an array of nanoliter volume sample sites, wherein thesample sites are through-hole wells with hydrophilic interiors andopenings surrounded by hydrophobic material.

One specific commercial example of a nanoliter chip system is theThru-Hole™ Array Technology made by Biotrove, Inc. of Woburn, Mass.Nanoliter chip technology relies on the ability to handle very smallvolumes of fluid samples, typically, less than 1000 nanoliters. Thevarious considerations taken into account in handling such small liquidsamples are known as microfluidics. A typical schematic of amicrofluidic array is shown in FIG. 1.

In FIG. 1, the sample wells 12 may be grouped into sub-arrays such as bycontrolling the spacing between the wells. For example, FIG. 2 shows achip 10 in which the sample wells 12 are grouped into a 4-by-12 array of5-well by 5-well sub-arrays 20. In another embodiment, the sub-arrays 20may be 8-wells by 8-wells or any other convenient number. The chip 10 inFIG. 2 is 1″×3″ to correspond to a standard microscope slide. The samplewells 12 in a sub-array 20 may be laid out in a square or rectangulargrid arrangement as shown in FIG. 2, or the rows and/or columns ofsample wells may be offset as shown in FIG. 1. The inter- and intra-gridthrough-hole spacing is precise to within less than ⅕ of a holediameter. For example, the through-holes in one embodiment of theBioTrove array are 320 micrometers in diameter with a center-to-centerspacing of 500+−25 micrometers.

The sample chip 10 typically may be from 0.1 mm to more than 10 mmthick; for example, around 0.3 to 1.52 mm thick, and commonly 0.3 mm.Typical volumes of the through-hole sample wells 12 could be from 0.1picoliter to 1 microliter, with common volumes in the range of 0.2-100nanoliters, for example, about 30 nanoliters. This corresponds to athrough-hole diameter of 350+−25 micrometers. Capillary action orsurface tension of the liquid samples may be used to load the samplewells 12. For typical chip dimensions, capillary forces are strongenough to hold liquids in place. Chips loaded with sample solutions canbe waved around in the air, and even centrifuged at moderate speedswithout displacing samples.

To enhance the drawing power of the sample wells 12, the target area ofthe receptacle, interior walls 42, may have a hydrophilic surface thatattracts a sample fluid. It is often desirable that the surfaces bebio-compatible and not irreversibly bind biomolecules such as proteinsand nucleic acids, although binding may be useful for some processessuch as purification and/or archiving of samples. Alternatively, thesample wells 12 may contain a porous hydrophilic material that attractsa sample fluid. To prevent cross-contamination (crosstalk), the exteriorplanar surfaces 14 of chip 10 and a layer of material 40 around theopenings of sample wells 12 may be of a hydrophobic material such as amonolayer of octadecyltrichlorosilane (OTS). Thus, each sample well 12has an interior hydrophilic region bounded at either end by ahydrophobic region.

The through-hole design of the sample wells 12 avoids problems oftrapped air inherent in other microplate structures. This approachtogether with hydrophobic and hydrophilic patterning enable self-meteredloading of the sample wells 12. The self-loading functionality helps inthe manufacture of arrays with pre-loaded reagents, and also in that thearrays will fill themselves when contacted with an aqueous samplematerial.

It has been suggested that such nanoliter chips can be utilized formassively parallel assays such as Polymerase Chain Reaction (PCR) andEnzyme-Linked Immunosorbent Assay (ELISA) analysis. However, nanoliterchips require complex time-consuming preparation and processing. Beforethe samples are introduced, each sample well must be pre-formatted withthe necessary probes, reagents, and other components in a processreferred to as formatting. Once the chip is formatted, the analyte orspecimen is introduced into each well, (sample loading), referringgenerically to both specimens loading and reagents loading. Transferringlarge collections of fluid samples such as libraries of small moleculedrug candidates, cells, probe molecules (e.g., oligomers), and/or tissuesamples stored in older style 96- or 384-well plates into more efficienthigh density arrays of nanoliter receptacles can be difficult. As apractical matter, there tend to be two approaches to formatting andloading of nanoliter sample chips—bulk transfer and discrete transfer.

An example of bulk transfer is dipping a sample chip into a reservoir ofsample liquid. The sample liquid wicks into the sample wells bycapillary action and all of the wells fill uniformly with the sample.

One method for discrete transfer uses a transfer pin loaded with thetransfer liquid. For example, pins or arrays of pins are typically usedto spot DNA samples onto glass slides for hybridization analysis. Pinshave also been used to transfer liquids such as drug candidates betweenmicroplates or onto gels (one such gel system is being developed by acompany in California). Many pin types are commercially available, ofvarious geometries and delivery volumes. Some are slotted, grooved,cross-hatched, or other novel-geometry pins. The Stealth Pin by ArrayItis capable of delivering hundreds of spots in succession from one sampleuptake, with delivery volumes of 0.5 nL to 2.5 nL. Majer PrecisionEngineering sells pins having tapered tips and slots such as theMicroQuil 2000. Example techniques for using one or more pins totransfer sample liquid are described in U.S. Patent Publication Number2003/7748 A1, filed Aug. 23, 2002, and incorporated herein by reference.

Due to the small dimensions involved, registration of the pin array tothe through-hole wells of the microfluidic array is non-trivial. Anymisalignment of the pins to the through-holes on the chip will result ina failure to properly load the microfluidic array with sample.

SUMMARY OF THE INVENTION

In accordance with one aspect of the invention, a method of registeringa location of a dispenser array in relation to a microfluidic array isprovided. A first one of the dispenser array and the microfluidic arrayis movable in relation to the frame, and the other of the first one ofthe dispenser array and the microfluidic array is fixed relative to theframe. The relative position of the dispenser array is identified by aset of coordinates. A first camera is in rigid association with one ofthe dispenser array and the microfluidic array. The method includesidentifying a fiducial reference in rigid association with the other oneof the dispenser array and the frame, in a manner permitting thefiducial reference to appear in a first position of a field of view ofthe first camera when the dispenser array is in an alignment position,associated with a first coordinate set, relative to the frame.Quantities related to a vector displacement from the alignment positionto a fixed position on the microfluidic array are determined. Thequantities thus determined are used to guide positioning of thedispenser array relative to the microfluidic array.

In accordance with related embodiments of the invention, the first oneof the dispenser array and the microfluidic array may be configured tomove independently in each of three approximately mutually orthogonaldirections. A skew from orthogonality of the directions may bedetermined. Determining skew may include positioning a plate in a fieldof view of the first camera, the plate having three reticules definingthree corners of a triangle. The first one of the dispenser array andthe microfluidic array may be moved relative to the frame so that afiducial reference appears in a plurality of distinct additionalpositions in the field of view of the first camera, each positionassociated with a distinct coordinate set. An orientation of the firstcamera relative to the directions may be determined based on theplurality of distinct positions.

In accordance with further related embodiments of the invention, asecond camera may be mounted in rigid association with the other one ofthe dispenser array and the microfluidic array. A second fiducialreference capable of being viewed by both the first camera and thesecond camera may be identified. The second fiducial reference may beviewed with both the first camera and the second camera to determinequantities of a vector displacement from a position within the field ofview of the first camera to a position within the field of view of thesecond camera.

In accordance with still further related embodiments, the first one ofthe dispenser array and the microfluidic array may be moved relative tothe frame so that a second fiducial reference on the one of thedispenser array and the microfluidic array appears in a plurality ofdistinct positions in the field of view of the second camera, eachposition associated with a distinct coordinate set. The orientation ofthe second camera relative to the directions may be determined based onthe plurality of distinct positions.

In accordance with yet another related embodiment of the invention, thefirst camera is in rigid association with the first one of the dispenserarray and the microfluidic array, the first camera being rigidlydisplaced from a fixed position on the first one of the dispenser arrayand the microfluidic array by a displacement vector. The method furtherincludes moving the first one of the dispenser array and themicrofluidic array relative to the frame so that the fixed position onthe first one of the dispenser array and the microfluidic array iswithin the field of view of the one of the second camera in rigidassociation with the frame. Quantities of the displacement vector aredetermined.

In accordance with other embodiments of the invention, a WorldCoordinate System having a center of origin is identified. A FirstCamera Coordinate System having a first camera center of origin within afield of view of the first camera is identified. A Second CameraCoordinate System having a second camera center of origin within a fieldof view of the second camera is identified. A transformation fortransforming a coordinate in the First Camera Coordinate System to theWorld Coordinate System is determined. A second transformation fortransforming a coordinate in the Second Camera Coordinate System to theWorld Coordinate System is determined.

In accordance with still further related embodiments, at least one ofthe dispenser array and the microfluidic array is rotatable around acenter of rotation. A position of the center of rotation is determined.Determining the coordinates of the center of rotation may includeapplying a best fit circle algorithm or another geometric algorithm.

In accordance with another aspect of the invention, a method ofregistering a dispenser array to a microfluidic array rigidly coupled toa frame is presented. The dispenser array is movable in relation to theframe. A first camera is in rigid association with the dispenser array,and a second camera is in rigid association with the frame. A set ofcoordinates identifies the relative position of the dispenser array. Themethod includes determining a first position of a first fixed point onthe microfluidic array relative to a fiducial reference by viewing themicrofluidic array with the first camera. A second position of a secondfixed point on the dispenser array relative to the fiducial reference isdetermined by viewing the dispenser array with the second camera.Quantities of a vector displacement from the first position to thesecond position are determined. The quantities thus determined are usedto guide positioning of the dispenser array relative to the microfluidicarray.

In accordance with related embodiments of the invention, a WorldCoordinate System having a center of origin is defined. A First CameraCoordinate System having a center of origin within a field of view ofthe first camera is defined. A transformation for transforming acoordinate in the First Camera Coordinate System to the World CoordinateSystem is determined. The World Coordinate System may include a firstaxis and a second axis orthogonal to the first axis, wherein thedispenser array is capable of moving along a first motion axis and asecond motion axis. The first motion axis is parallel to the first axis,while the second motion axis defines an angle β with the second axis.The angle β is determined. Calculating the angle β may includepositioning a plate in a field of view of the first camera, the platehaving three reticules defining three corners of a triangle and/orrectangle. The first camera coordinate system may include a camera firstaxis and a camera second axis orthogonal to the camera first axis, thecamera second axis defining an angle θ_(C) with the second motion axisof the first camera, the method further comprising calculating the angleθ_(C). Calculating the angle θ_(C) may include viewing a fiducialreticle in the field of view of the first camera; moving the firstcamera along only the x motion axis; and moving the first camera alongonly the y motion axis.

In accordance with further related embodiments of the invention, asecond camera is in rigid association with the frame. The method furtherincludes defining a Second Camera Coordinate System having a secondcamera center of origin within a field of view of the second camera. Asecond transformation for transforming a coordinate in the Second CameraCoordinate System to the World Coordinate System is determined.

Determining the second transformation may include positioning thedispenser array in the field of view of the second camera, and movingthe dispenser array along the x-motion axis. Determining the secondtransformation may include determining the second camera center oforigin in the World Coordinate System. Determining the second cameracenter of origin in the World Coordinate System may include placing afiducial in a field of view of the second camera. The fiducial is movedsuch that the fiducial is in the field of view of the first camera. Thecoordinates of the fiducial in the First Camera Coordinate System aredetermined. The coordinates of the fiducial in the First CameraCoordinate System are transformed to the World Coordinate System. Thesecond camera may then be used to determine the coordinates of thedispenser array in the World Coordinate System.

In accordance with yet other embodiments of the invention, the dispenserarray is rotatable around a center of rotation in rigid association withthe first camera. The method further includes determining thecoordinates of the center of rotation in the World Coordinate System.The coordinates of the center of rotation in the World Coordinate Systemmay be determined by rotating the dispenser array and the microfluidicarray in a field of view of the second camera and using a best fitcircle algorithm or another geometric algorithm. A vector between thefirst camera and the center of rotation may be determined, the vectorhaving coordinates in the World Coordinate System. The dispenser arraymay include a center and define a reference axis. The reference axisdefines an angle θ_(RA) with an axis of the World Coordinate System. Thecoordinates of the center and the angle θ_(RA) are determined.

More specifically, θ_(RA) may be determined by looking at each dispenserof the dispenser array using the second camera. The dispensers mayapproximately form a grid, thus a grid fit algorithm may be used. Thegrid fit algorithm may return the center of the grid as well as theangle of the grid. The center of the grid may not be the center ofrotation. Various algorithms may be in defining the grid fit. Onealgorithm that may be used minimizes the average amount of error betweenthe real grid and the fitted grid. Another algorithm may minimize theworst case error of any pin on the real grid to the fitted grid.

In accordance with still further embodiments of the invention, thecoordinates of the microfluidic array in the World Coordinate System aredetermined. Determining the coordinates of the microfluidic array in theWorld Coordinate System may include moving the first camera so as toplace the some or all of the microfluidic array field in the field ofview of the first camera. For example, the four corners sample wells ofthe microfluidic array/chip may be viewed to determine where therectangle of the chip is defined and the rotation of the chip. Invarious embodiments, the entire microfluidic array/chip may be placed inthe field of view of the camera.

In accordance with another aspect of the invention, a system forregistering a location of a dispenser array in relation to amicrofluidic array includes a first camera in rigid association with oneof the microfluidic array and the dispenser array. A frame is rigidlycoupled to the other one of the microfluidic array and the dispenserarray, the one of the microfluidic array and the dispenser movable inrelation to the frame.

In accordance with related embodiments of the invention, a second camerais in rigid association with the frame, the second camera capable ofviewing the first camera and the one of the microfluidic array and thedispenser array. A controller may control relative motion between themicrofluidic array and the dispenser array. The controller may includeat least one encoder and/or an interface to the first camera fortransferring data signals. A rotary stage may rotate one of themicrofluidic array and the dispenser array. A gantry may be used tocontrol motion of the dispenser array relative to the microfluidicarray, for example, in three approximately mutually orthogonaldirections.

In accordance with another embodiment of the invention, a method ofaligning a first array to a second array is provided. Each elementwithin the first array has an expected position relative to otherelements in the first array. The expected positions of each element inthe first array form a template, wherein one or more elements of thefirst array deviate from their expected position. The method includesaligning the template with the first array. The template is used toapproximate locations of each element in the first array. Elements ofthe first array are aligned with elements the second array as a functionof the approximate locations.

In related embodiment of the invention, aligning the template with thefirst array includes aligning at least one position on the template withat least one position on the first array. A camera may be used todetermine the location of the at least one position on the first array.Using the camera may include viewing segments of the first array todetermine the location. Alternatively, the entire first array may beviewed simultaneously.

In further related embodiments, one of the first array and the secondarray may include receptacles, and the other of the first array and thesecond array includes pins. The pins may have a pin diameter, and eachreceptacle may define an opening having a diameter that is larger thanthe pin diameter by a predefined value. Thus, when the elements of thefirst array are aligned with the second array as a function of theapproximate locations, a pin that deviates from their expected positionby less than the predefined value is still aligned over theircorresponding receptacle. The pins may be cone shaped. Each of the pinsmay be capable of limited movement. The receptacles may have wallsdefining a beveled opening. The receptacles may be through-holes, themethod further including viewing the pins through the through-holesusing a camera.

In yet other related embodiments of the invention, each element withinthe second array has an expected position relative to other elements inthe second array, the expected positions of each element in the secondarray forming a second template, wherein one or more elements of thesecond array deviate from their expected position. The method furtherincludes aligning the second template with the second array. Thetemplate is used to approximate locations of each element in the secondarray. Elements of the first array are aligned with the second array asa function of the approximate locations of each element in the secondarray. The first template may be substantially equivalent to the secondtemplate.

In another embodiment of the invention, a method of dispensing amicrofluidic sample is provided. The method includes providing an arrayof dispensers for dispensing the microfluidic sample, and an array ofreceptacles, each receptacle capable of receiving microfluidic samplefrom one of the dispensers. At least one of the array of dispensers andthe array of receptacles is scanned with a camera for a fiducialreference. The array of dispensers is aligned with the array ofreceptacles as a function of the fiducial reference such that transferof sample from the array of dispensers to the array of receptacles isenabled.

In related embodiments of the invention, each dispenser and/orreceptacle has an expected position relative to other dispensers in thedispenser and/or receptacle array. The expected positions of eachdispenser and/or receptacle in the dispenser and/or receptacle arrayforming a template, wherein one or more of the dispensers and/orreceptacles deviate from their expected position. The method furtherincludes aligning the template with the dispenser and/or receptaclearray based on the fiducial position. The template is used toapproximate locations of each element in the dispenser and/or receptaclearray. The elements of the dispenser array are aligned with thereceptacle array as a function of the approximate locations.

In further related embodiments of the invention, scanning includes usingthe camera to view segments of at least one at least one of the array ofdispensers and the array of receptacles to determine the fiducialposition. Alternatively, the camera may simultaneously view the entireat least one of the array of dispensers and the array of receptacles todetermine the fiducial position.

In still further related embodiments of the invention, the array ofdispensers may be an array of transfer pins for transferring a pluralityof samples to a corresponding plurality of the receptacles. The pins maybe cone shaped. Each of the pins may be individually capable of limitedmovement. The receptacles may have walls defining a beveled opening. Thearray of receptacles may be a platen array of through-hole wells. Thedispensers may be viewed through the through-hole wells using thecamera. The array of receptacles may be a platen array of closed-endedwells. At least one of the receptacles may include hydrophilic wallsthat attract the sample. At least one of the receptacles may include anopening surrounded by hydrophobic material.

In accordance with another embodiment of the invention, a system fordispensing sample fluid includes one or more dispensers forming adispenser array. At least one receptacle array includes one or morereceptacles; each receptacle capable of receiving sample fluid from oneof the dispensers. An alignment means aligns the dispenser array and theat least one receptacle array.

In accordance with related embodiments of the invention, the alignmentmeans includes a rotational stage for rotating at least one of thedispenser array and the at least one receptacle array. The alignmentmeans may include a vision means for viewing one at least one of thedispenser array and the at least one receptacle array. The vision meansincludes one or more sensors for detecting position of at least one ofthe dispenser array and the receptacle array, which may be one of aoptic sensor and an acoustic sensor. The vision means may be a camera.One of the dispenser array and the receptacle array may include afiducial reference, the vision means capable of viewing the fiducialreference to align the dispenser array and the at least one receptaclearray.

In accordance with further related embodiments of the invention, the atleast one receptacle array may be one of a platen and a biochip. Thereceptacle array may be a microfluidic array. The receptacles mayinclude closed and/or through-hole wells. The at least one receptaclearray may include a plurality of receptacle arrays positioned in a tray.The dispenser array may be an array of transfer pins for transferring aplurality of samples to a corresponding plurality of the receptacles.The pins may be cone shaped and/or individually capable of limitedmovement. The receptacles may have walls defining a beveled opening. Theat least one of the receptacles may include hydrophilic walls thatattract the sample. The at least one of the receptacles may include anopening surrounded by hydrophobic material.

In accordance with still further embodiments of the invention, eachelement within the dispenser array may have an expected positionrelative to other elements in the dispenser array, the expectedpositions of each element in the dispenser array forming a template,wherein one or more elements of the first array deviate from theirexpected position. The alignment means may include template means foraligning the template with the dispenser array; approximating locationsof each element in the dispenser array using the template; and aligningelements of the first array with the second array as a function of theapproximate locations.

Other various embodiments relate to differentially coated plates,methods for preparing said plates, and methods for using thedifferentially coated plates for conducting a plurality of specifiedreactions. Thus, for example, some embodiments provide a process fordifferentially treating surfaces of a substrate, such as channelsubstrate or a through-hole array plate, wherein the different surfacesare subjected to a series of processes and reactions which result indifferential surface coatings for the etched or through-hole surface andthe surface of the substrate or plate. A differential coating is acoating or process whereby the coating physicochemical properties varyin a controlled manner, and in a controlled pattern. These coatingproperties can be surface tension, reactivity, biocompatibility, andnumerous other properties. This is different from thehydrophobic/hydrophilic patterning of a planar surface, as in the caseof a MALDI target plate where small hydrophilic spots are surrounded bylarge hydrophobic areas to concentrate the sample onto the hydrophilicspot during drydown. In the case of the OpenArray™ plate, the inside ofthe through-holes is preferably hydrophilic and biocompatible, whereasthe outside surface of the plate is preferably hydrophobic andnon-protein binding, although it is envisioned that properties for theinside surface relative to the outside surface may change, as needed.When an OpenArray™ chip is differentially coated such that thethrough-holes are hydrophilic and the exterior of the chip ishydrophobic, a chip may be dipped into a high surface tension fluid suchas water, and when removed, the individual through-holes are filled withfluid and the exterior of the chip is dry.

The following provides generalized methods and devices for spatiallycontrolling the surface modifications of an etched surface and is afocus of this invention. One important aspect of the invention is theability to force load liquids into the OpenArray™ for exchanging fluidsthat enable the differential coating process. During the manufacture ofthe OpenArray™ chips, they become uniformly hydrophobic. When loading isattempted to perform chemistry in the through-holes to affectdifferential coating, a high surface tension fluid will not load thethrough-holes, and a low surface tension fluid will wet the entire chip.It becomes difficult to perform differential coating a simple way.Therefore, the novel technique of forced loading was invented. Theuniformly hydrophobic chip is submerged in a low surface tensionco-solvent, such as ethanol. Then the chip is submerged in a highsurface tension fluid that contains reagents for the differentialchemistry. The high surface tension fluid exchanges and replaces the lowsurface tension fluid in the wells. When the chip is removed from thehigh tension fluid, the high surface tension fluid sheets off theexterior coating of the chip, but remains in the through-holes. If thedifferential coating takes time to complete, and evaporation of the highsurface tension fluid from the through-holes is problematic, the chipcan then be submerged in an immiscible fluid such as silicone oil or aperfluorinated hydrocarbon. This keeps the high surface tension fluid inthe wells while the chemistry or deposition occurs, without evaporationor leakage of the high surface tension fluid onto the exterior of thechip.

This process can be generalized beyond hydrophilic interior andhydrophobic exterior coatings to, for example, reverse surface tensionsof the liquids relative to the platen substrate to make the interiorsurface hydrophobic and the exterior surface hydrophilic. This would beuseful in loading polar solvents, such as benzene, into the OpenArraychannels for non-biological chemistry applications.

Methods exist for the aqueous treatment or aqueous surface chemistrymodification of substrates containing hydrophobic pores. For example,U.S. Pat. No. 5,209,850, assigned to Gore, teaches the use of lowsurface tension co-solvents such as methanol or surfactants to allowwetting of the substrate and subsequent surface modification. However,these methods uniformly treat the entire substrate and do not providedifferential coatings.

We have developed a number of techniques and embodiments that allowdifferential coating of the OpenArray™ chip, and could also be appliedto other substrates with hydrophobic pores. There are severalgeneralized approaches for achieving a differentially coated etchedsubstrate:

Deposition of one material (e.g. hydrophobic coating) on exterior whileblowing a non-reactive gas (N₂) through the through-holes to block itsdeposition on the interior surfaces of channel. Second material isloaded, if needed, as a liquid into the channels to react and chemicallyor physically modify and coat the interior surface. The second materialdoes not wet nor react with the first material to change itsphysicochemical properties.

Deposition of one material (e.g. hydrophobic coating) on exterior whilephysically blocking through-holes with a liquid or solid. Unblockingholes without affecting hydrophobic coating and then, if needed, coatingchannel interiors with a different material (e.g. hydrophilic).

Uniform deposition of one material (e.g. hydrophobic coating) onthrough-hole array. Subsequent stripping of coating from insidethrough-holes by the use of a mask and high energy UV light to exposesubstrate. Second material deposited with process gas or liquid oninterior of through-holes. UV light may/may not be used to initiatechemical reaction for deposition/chemical modification of interiorsurfaces.

Uniform deposition of one material (e.g. hydrophobic coating) onthrough-hole array. Subsequent stripping of coating from thethrough-holes by forced loading of an etching solution thatphysiochemically removes the first material from the interior surfacesof the channels. Second material for coating/modifying interior surfacesis applied by liquid or gas deposition.

Individually addressing the through-holes of a uniformly treated chip(e.g. hydrophobically) with a dispenser, such as a pin, a syringe tip, apiezo dispenser, etc. to deposit an etchant or other liquid that reactswith the first material to product a surface with the desired properties(e.g. hydrophilic). This could be a multi-step process involving firstapplying an etchant to remove the hydrophobic coating, a cleaning agentto clean and activate the surface and the hydrophilic coating.

Using optical masks and UV-catalyzed chemistry to cure differentcoatings on the interior surfaces on the through-holes and the chipexterior. The patterned substrate may be a stainless steel plate havingetched grooves or patterns, or a stainless steel plate havingthrough-holes such as a microfluidic sample array (a chip). In otherembodiments, the patterned substrate may be of silicon or glass. Variousmethods of differentially treating the patterned substrate orthrough-hole array plate may include a series of treatments andreactions. The series of treatments in various embodiments in accordancewith the presently claimed invention may involve inspection; labelingfor tracking during processing; cleaning; uniform coating of the planarnon-etched or non-holed substrate surface with a first reagent; treatingthe etched surface or through-hole surface with a reagent that activatesthe etched surface or through-hole surface for later treatment byproducing reactive groups on the etched or through-hole surfaces;treating the etched or through-hole surfaces with a reagent differentfrom that used to treat the planar non-etched or non-holed surface ofthe substrate; additional treatment of the etched or through-holesurface to prevent reaction with reagents which may be used in latertreatments; coating the planar non-etched or non-holed surface of thesubstrate with a second reagent that either adsorbs or chemically reactswith the first reagent on the non-etched or non-holed surface; andquality control tests.

A particular embodiment may first uniformly coat all surfaces of thesubstrate by treating with vinyl-terminated silane, for example, thenselectively-oxidize the etched or through-hole surfaces by force loadingwith ethanol a permanganate solution, for example, of 5 mM KMnO₄ and19.5 mM NaTO₄ in deionized water followed by incubation in a nonreactiveoil or liquid such as a perfluorinated alkane solution for 2 hours, thenPEGylation (covering with polyethylene glycol or molecules bearing PEGmoieties) of the selectively oxidized etched or through-hole surface isdone followed by re-loading of the etched or through-holed surface withadditional PEG, with a final coating of the non-etched or non-holedsurface accomplished by treatment with perfluorosilane using vapor-phasedeposition.

Alternatively, the initial coating of all surfaces of the substrate maybe done using liquid phase vinyl deposition with7-octenyltrichlorosilane or 10-undecenyltrichlorosilane, for example.The PEGylation may alternatively be performed using various PEG-silanederivatives, such as methoxy-PEG silane MW 2000, methoxy-PEG silane MW500, and methoxy-PEG silane MW 10,000. Also, triethoxysilylbuteraldehydemay be used as an alternative first coating of the non-etched ornon-holed planar surface of the substrate. This allows PEGylation of theetched or through-hole surface without selective oxidation. OtherPEGylation methods include the use of longer PEG molecules, such asmethoxy-PEG-amine MW 5000 in place of silane-PEG coatings of lowermolecular weights. In addition, the PEG within the PEGylated etched orthrough-hole surfaces may be cross-linked using hyperbranched PEG andPEI molecules and appropriate cross-linker molecules.

In such embodiments, the etched or through-hole surface is treated witha reagent to expose functional group A, followed by exposure to asolution containing a PEG having a terminal functional group B that isreactive with functional group A. The OpenArray could be coated withdifferent materials catalyzed by absorption of radiant energy (UV,X-ray, electron beam) at different locations in the array by one or moreof the previously described embodiments and then by use of one or moremasks, different materials could be formed in different regions of theOpenArray by exposure to radiation for specified durations, intensitiesand energies (e.g. UV, X-ray, electron beam). Instead of masks, theradiation could be focused and directed to specified locations by anumber of focusing means including lenses. The focused beam (photons orelectrons) is moved relative to the platen to trace a prescribed patternof surface modifications. The optical based method can be combined withone or more of the previous embodiments for applying a differentialcoating to an OpenArray.

Other embodiments provide a method for differentially coating asubstrate having at least one exterior surface and a plurality ofchannels for liquid disposed therein, each channel having at least oneinterior surface in communication with at least one exterior surface,the method comprising applying a first coating agent to the substrate tocreate a coated substrate and selectively modifying at least oneexterior surface or at least one interior surface of the coatedsubstrate with a first modifying agent to create at least one modifiedsurface having a first specified physicochemical property and anon-modified surface such that the modified surface differs from thenon-modified surface with respect to the first specified physicochemicalproperty. In some embodiments, a second modifying agent is applied tothe first modified surface such that the second modifying agent impartsa second specified physicochemical property to the modified surface. Inrelated embodiments, the first modifying agent may be applied in amixture of two or more modifying agents having distinct specifiedphysicochemical properties, to create at least one modified surfacehaving a mixture of distinct specified physicochemical properties.

According to this method, the modified surface may be a modifiedgradient surface having a gradient mixture of the distinct specifiedphysicochemical properties, or the modified surface may be a modifiedmixed-layer surface having a heterogeneous mixture of distinct specifiedphysicochemical properties. Alternatively a third or more modifyingagent may be applied to the modified agent to impart a third or morespecified physicochemical property to the modified surface. Otherrelated embodiments provide a variation of the described method whereinthe at least one modified surface has one or more of the first, secondand third specified physicochemical properties or it may have acombination of the specified physicochemical properties.

Related embodiments provide methods where the modifying agent is applieduniformly to create at least one uniformly modified surface, or appliednon-uniformly such that the non-uniformly modified surface isnon-uniform with respect to a property selected from the groupconsisting of concentration of modification, thickness of modification,continuity of modification, presence of the specified physicochemicalproperty, and nature of the specified physicochemical property.

Another embodiment in accordance with the present invention provides amethod as described above, wherein selectively modifying furthercomprises selectively activating at least one interior surface or atleast one exterior surface with an activating agent prior to selectivelymodifying said surface so as to create at least one activated surfaceand modifying the at least one activated surface by reacting the surfacewith the modifying agent to create at least one modified surface withthe first specified physicochemical property. In some embodiments, theexterior or the interior surface is modified. In related embodiments,the method further comprises causing a blocking agent to adhere to atleast one interior surface or at least one exterior surface prior toselectively applying the first coating agent so as to create at leastone blocked surface, and removing the blocking agent from the blockedsurface after applying the first coating agent. The blocked surface mayeither be at least one exterior surface, or at least on interiorsurface.

In other more particular embodiments there is provided a method asdescribed above, wherein the method further comprises causing a blockingagent to adhere to at least one modified surface to create at least oneblocked modified surface, applying a second coating agent to thesubstrate and removing the blocking agent from the blocked modifiedsurface. As in other embodiments, the modified surface may be anexterior surface or an internal surface and the channels may bethrough-holes, passages or troughs.

In other particular embodiments the physical interactions between theblocking agent and the surface are selected from one or more of thegroup consisting of Van der Waal's forces, hydrogen bondinginteractions, hydrophilic interactions, hydrophobic interactions,dipole-dipole interactions, ionic interactions, surface tensioninteractions, and polar interactions. Alternatively, the coating agentor agents, blocking agents, and/or first and later modifying agentsadhere to the exterior or interior surface by chemical bonding such ascovalent bonding, coordinate covalent bonding, or ionic bonding.

Still other embodiments provide a method for differentially coating asurface wherein the first, second and other modifying agents areselected from the group consisting of a polymer, a monomer, across-linking agent, a photo-cleavable agent, a silane, a lipid, a fattyacid, an amino acid, a peptide, a protein, an antibody, an enzyme, asurfactant, a micelle, a liposome, a nucleotide, an oligonucleotide, anucleic acid, a salt, a wax, a low-melting solid, and an oil. In thisand other related embodiments, the first and second coating agents mayalso be selected from the group consisting of a polymer, a monomer, across-linking agent, a photo-cleavable agent, a silane, a lipid, a fattyacid, an amino acid, a peptide, a protein, an antibody, an enzyme, asurfactant, a micelle, a liposome, a nucleotide, an oligonucleotide, anucleic acid, a salt, a wax, a low-melting solid, and an oil. Andaccording to the desired type of differentially coated device desired,the blocking agent may be selected form the group consisting of apolymer, a monomer, a cross-linking agent, a photo-cleavable agent, asilane, a lipid, a fatty acid, an amino acid, a peptide, a protein, anantibody, an enzyme, a surfactant, a micelle, a liposome, a nucleotide,an oligonucleotide, a nucleic acid, a salt, a wax, a low-melting solid,and an oil.

In some particular embodiments, selectively modifying a surface mayfurther comprise applying a solvent having a lower surface energy thanthat of the modifying agent to the at least one exterior surface or theat least one interior surface prior to applying the modifying agent, orselectively modifying may further comprise subjecting the substrate to apressure differential prior to applying the modifying agent or bysubjecting the substrate to a liquid stream of modifying agent such thatmomentum from the liquid stream applies the modifying agent to the atleast one interior surface.

In a related particular embodiment there is provided a method fordifferentially coating a device wherein selectively modifying furthercomprises applying the modifying agent by exposing at least one exteriorsurface to a reactive vapor while subjecting at least one interiorsurface to a laminar flow stream of non-reactive gas such that the gasstream prevents the reactive vapor molecules from interacting with thatinterior surface.

Some embodiments provide that the activator, if used, as an oxidizingagent, a cross-linking agent, or an energy source, and may be a solid ofa liquid such that the blocking agent makes at least one blocked surfaceany one or more of impermeable to electrons; impermeable to ionizingradiation; or unavailable for or resistant to further chemical reaction,light-activated reaction, or chemical modification. Moreover, inparticular embodiments the blocking agent is selected from the groupconsisting of a polymer, a monomer, a cross-linking agent, aphoto-cleavable agent, a silane, a lipid, a fatty acid, an amino acid, apeptide, a protein, an antibody, an enzyme, a surfactant, a micelle, aliposome, a nucleotide, an oligonucleotide, a nucleic acid, a salt, awax, a low-melting solid, and an oil.

In a more particular embodiment there is provided a method fordifferentially coating a substrate having at least one exterior surfaceand a plurality of channels for liquid disposed therein, each channelhaving at least one interior surface in communication with at least oneexterior surface, the method comprising applying a first coating agentto the substrate to create a coated substrate, blocking at least oneexterior surface or at least one interior surface of the coatedsubstrate to create at least one blocked surface and at least onenon-blocked surface, selectively modifying the non-blocked surface witha modifying agent to create at least one modified surface and at leastone non-modified surface such that the modified surface differs from thenon-modified surface with respect to a specified physicochemicalproperty, and unblocking the at least one blocked surface. The blockedsurface may be an exterior surface or an interior surface, andselectively modifying comprises exposing the at least one non-blockedsurface to UV radiation, ionizing radiation, an ion-beam, an electronbeam, microwave radiation, visible light, a coherent light source, or alaser such that the specified physicochemical property is achieved. Inalternative embodiments, selectively modifying comprises treating the atleast one non-blocked surface with an etching material that removes thecoating material from the non-blocked surface.

Related embodiments provide methods for differentially coating asubstrate, wherein applying the first coating agent comprises uniformlyor non-uniformly applying the coating agent, such that the first coatingagent is one or more of a hydrophobic agent, a hydrophilic agent or amultifunctional agent and the specified physicochemical property of theat least one modified surface is a hydrophobic property or a hydrophilicproperty. In other related embodiments, applying the second coatingagent comprises uniformly or non-uniformly applying the coating agentand the second coating agent may be one or more of a hydrophobic agent,a hydrophilic agent or a multifunctional agent wherein the specifiedphysicochemical property of the at least one modified surface is ahydrophobic property or a hydrophilic property.

Another particular embodiment provides a differentially coated devicefor conducting a plurality of nano-volume specified reactions, thedevice comprising a platen having at least one exterior surface modifiedto a specified physicochemical property, a plurality of nano-volumechannels, each nano-volume channel having at least one interior surfacein communication with the at least one exterior surface, wherein the atleast one interior surface of one or more of the of nano-volume channelsis selectively coated with an optionally dissolvable coating agentphysisorbed to the at least one interior surface, wherein the optionallydissolvable coating agent comprises a coating agent and a firstcomponent for the plurality of specified reactions. In embodimentshaving the dissolvable coating agent, the coating agent is selected fromthe group consisting of a low melting-point solid; a hydratable solid,semi-solid or gel, a polymer, a block co-polymer, a polyethylene glycol,a polyvinyl alcohol, a polyethyleneimine, a lipid, a surfactant, acontrolled-release agent, and a salt.

In related embodiments wherein a method for preparing a differentiallycoated device is provided, the method comprises providing a devicehaving a plurality of nano-volume channels for retaining one or morereaction components for the specified reactions, each channel having atleast one interior surface in communication with at least one exteriorsurface, selectively coating at least one interior surface of one ormore of the plurality of channels with a dissolvable coating agent so asto prepare the differentially coated device, wherein the dissolvablecoating agent is physisorbed to the at least one interior surface andcomprises a coating agent and a first component for the plurality ofspecified reactions.

Other embodiments provide a method for preparing a differentially coateddevice as described above, wherein providing further comprisesselectively coating at least one external surface with a coating agentto impart a specified physicochemical property, using an autoloaderdevice comprising a loading chamber sufficiently large to contain thedevice to be coated, the coating reagent, and an immiscible fluid thatlifts the coating reagent over the device as the volume of theimmiscible fluid is increased.

In such embodiments, as before, the coating agent is selected from thegroup consisting of a low melting-point solid; a hydratable solid,semi-solid or gel; a polymer; a block co-polymer; a polyethylene glycol;a polyvinyl alcohol; a polyethyleneimine; a lipid; a surfactant; acontrolled-release agent; and a salt, and loading further comprisesusing a pin-loading device comprising an array of calibrated transferpin dispensers to load the plurality of channels with one or morereaction mixtures, or using a piezoelectric disperser to load thechannels. In a related embodiment there is provided a method forconducting a plurality of specified reactions using the differentiallycoated device of above, the method comprising loading the plurality ofchannels in the differentially coated device with one or more reactionmixtures comprising additional components sufficient for the pluralityof specified reactions, and initiating the plurality of reactions by oneor more methods selected from the group consisting of heating thedevice, exposing the device to photo/optical conditions, hydrating thenano-volume channels; and stacking the device with one or more otherdevices to mix components in each device upon contact, thus initiatingthe specified reactions.

A more particular embodiment provides a method for conducting aplurality of nano-volume specified reactions as described, whereinloading further comprises using a pin-loading device comprising an arrayof calibrated transfer pin dispensers to load the plurality of channelswith one or more reaction mixtures, or using a piezoelectric disperserto load the channels. Related embodiments provide a plurality ofchannels that may vary from channel to channel with respect to any orall of: a specified reaction, a sample, a specified physicochemicalproperty of the nano-volume channel, and method of initiating aspecified reaction, wherein the plurality of channels comprise aplurality of nano-volume through-holes or nano-volume troughs andwherein the plurality of specified reactions comprise differentspecified reactions in two or more of the plurality of channels. Inother embodiments, the plurality of specified reactions may comprise thesame specified reaction in two or more of the plurality of channels, andmay comprise maintenance of cell cultures. U.S. Pat. No. 6,716,629 toHess et al. filed Oct. 1, 2001 and US Published Applications 20030180807A1 and 20030124716 A1 to Hess et al. both filed Dec. 10, 2002 disclosemethods for maintaining cell cultures in arrays, and apparatus for same,each of which is hereby incorporated by reference herein in theirentirety.

Another particular embodiment provides a method for preparing adifferentially coated device for conducting a plurality of nano-volumespecified reactions, the method comprising providing a device having aplurality of channels for retaining one or more reaction components forthe specified reactions, each channel having at least one interiorsurface in communication with at least one exterior surface, selectivelycoating at least one interior surface of the plurality of channels witha dissolvable coating agent so as to prepare the differentially coateddevice, or wherein the dissolvable coating agent is physisorbed to theat least one interior surface and comprises a coating agent andoptionally a first component for the plurality of specified reactions.In related embodiments, selectively coating further comprises using apin-loading device comprising an array of calibrated transfer pindispensers to selectively physisorb the dissolvable coating agent to theat least one interior surface. Alternatively, the loading device may bepiezoelectric disperser.

Still another embodiment provides a method for conducting a plurality ofnano-volume specified reactions using a first and at least seconddifferentially coated device prepared described above, the methodcomprising loading the plurality of channels in the first differentiallycoated device with one or more samples, wherein the samples are the sameor different, initiating the plurality of reactions by stacking thefirst device with at least a second differentially coated device loadedwith a mixture of reagents comprising sufficient components common tothe specified reactions, such that upon stacking, the mixture ofreagents in the at least second device mixes with the samples of thefirst device, thereby initiating the specified reactions. In relatedembodiments, loading further comprises using a pin-loading devicecomprising an array of calibrated transfer pin dispensers to load theplurality of nano-volume channels with one or more samples, or loadingfurther comprises using a piezoelectric disperser to load the channels.

Still another more particular embodiment provides a method for preparinga differentially coated device for conducting a plurality of nano-volumespecified reactions, the method comprising preparing a platen having aprescribed spatial arrangement of micro-volume channels, eachmicro-volume channel having at least one interior surface incommunication with at least one exterior surface, wherein preparingfurther comprises coating a flexible printing pad with a coating agent,wherein the coating agent is applied to the exterior surface to create aspatially arranged coating on the printing pad which correlates to theprescribed spatial arrangement for the channels, transferring thespatially arranged coating on the flexible printing pad to the platen bycontacting at least one external surface of the platen with the printingpad to impart the prescribed spatial arrangement of micro-volumechannels to the platen, and selectively coating the at least oneinterior surface of one or more of the plurality of micro-volumechannels with a coating agent so as to prepare the differentially coateddevice, wherein the coating agent is physisorbed to the at least oneinterior surface and comprises a coating agent and optionally a firstcomponent for the plurality of specified reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features of the invention will be more readily understoodby reference to the following detailed description, taken with referenceto the accompanying drawings, in which:

FIG. 1 shows a detailed cut away view of a typical nanoliter sample chipaccording to the prior art.

FIG. 2 shows a top plan view of a chip according to FIG. 1 in which thearray of sample wells is grouped into sub-arrays.

FIG. 3 shows a system for registering a dispenser array to amicrofluidic array 303, in accordance with an embodiment of theinvention.

FIG. 4 shows the rotary stage according to FIG. 3 in more detail.

FIG. 5 shows an illustrative process of registering a dispenser array toa microfluidic array, in accordance with an embodiment of the invention.

FIG. 6 shows relative positions of a World Coordinate System, a Firstcamera Coordinate System, and a dispenser array, in accordance with anembodiment of the invention.

FIG. 7 shows a fiduical reference that may be viewed in a plurality ofdistinct positions in the field of view of a first camera, in accordancewith an embodiment of the invention.

FIG. 8 shows a plate with three accurately known points that may be usedto determine angle β, in accordance with an embodiment of the invention.

FIG. 9 shows two reticles defining an angle θ_(R), in accordance with anembodiment of the invention.

FIG. 10 shows a vector T_(PC) formed between a first camera's O_(C) andthe center of rotation (O_(P)) of a dispenser array, in accordance withan embodiment of the invention

FIG. 11 shows a layout of an exemplary dispenser array, in accordancewith one embodiment of the invention.

FIGS. 12(a) and 12(b) shows four points found at the corners of amicrofluidic array that are used in determining the orientation of themicrofluidic array, in accordance with an embodiment of the invention.

FIG. 13 shows a system for registering a dispenser element to amicrofluidic array, in accordance with an embodiment of the invention.

FIG. 14 shows rotation of a microfluidic array, in accordance with anembodiment of the invention.

FIG. 15 shows a system for registering a dispenser array that has aplurality of dispenser elements with a microfluidic array, in accordancewith an embodiment of the invention.

FIG. 16 shows a depiction of one way to differentially coat a device inaccordance with an embodiment of the invention, wherein uni-directionalflowing fluorosilane gas is used to coat an exterior surface of thedevice, and opposite-flowing N₂ is used to protect the channels.

FIG. 17 shows a schematic of an alternative way to differentially coat adevice in accordance with an embodiment of the invention, whereinbi-directional flowing fluorosilane gas is used to coat two exteriorsurfaces of the device, such that the channels are protected from thesilane coating agent by a wax, which is dissolvably removed later bymelting.

FIG. 18 shows another a way to differentially coat a device inaccordance with the present invention, wherein the entire device,including the plurality of nano-volume channels, is first coated with ahydrophobic film—fluorosilane in this case—followed by blocking ormasking of on external surface, such that treatment with UV light stripsthe silane from the channels but not the masked/blocked externalsurface.

FIG. 19 shows a schematic of a method for differentially coating adevice in accordance with the present invention, wherein the entiredevice, including the nano-volume channels, is first coated with ahydrophobic film, such as fluorosilane, as depicted here. The channelsare then subjected to an etching/modifying agent such as strong base,which selectively removes the hydrophobic film from the nano-volumechannels.

FIG. 20 depicts yet another way to differentially coat a device inaccordance with the present invention, wherein the device is completelycoated with a reactive film, in this case a reactive silane, after whichfirst a hydrophilic and then second a hydrophobic film are applied onthe channel and external surfaces, followed by selective removal of thehydrophilic and second hydrophobic layers by subjecting the device,particularly the nano-volume channels, to 360-nm light which catalyzesthe release of the hydrophilic and subsequent hydrophobic layers fromthe surface of the channels, leaving only the original hydrophilic layerin the channels, and the other layers on the external surfaces.

FIG. 21 shows a method for differentially coating a device using contactprinting or stamping methods, in accordance with embodiments of theinvention, wherein a printing pad or stamp pad of coated with a firsthydrophobic agent namely polydimethylsiloxane (PDMS), then layered withoctadecyltriclorosilane (OTS). By contacting the device with theOTS-layered printing pad, at least one external surface of the device iscoated with the OTS, thus allowing controlled application of compoundsto the external surfaces of the device, such as OTS in from the printingpad to the device, after which the internal surfaces of the array devicemay be modified using reagents such as aldehyde-terminated silane andamine-terminated polyethylene glycol (PEG); and carboxyl-terminatedPEG+amine-terminated PEG.

FIG. 22 shows the use of forced loading to load channels in ahydrophobically coated device with ethanol, a lower energy (surfacetension) solvent, followed by immersion of the ethanol-loaded devicewith an aqueous solution, the lower surface tension ethanol “pulling”the higher energy (surface tension) water into the nano-volume channels,after which the device may be dipped in an aqueous solution to exchangewater for aqueous reagents, thereby enabling chemical reactions to beconducted in the nano-volume channels as the device is incubated in oil,for example

FIG. 23A shows the initial steps of a process for differentially coatingthe surfaces of a patterned substrate in accordance with an embodimentof the presently claimed invention.

FIG. 23B shows several additional steps of the same process fordifferentially coating the surfaces of a patterned substrate inaccordance with an embodiment of the presently claimed invention.

FIG. 23C shows three later steps of the same process for differentiallycoating the surfaces of a patterned substrate in accordance with anembodiment of the presently claimed invention.

FIG. 24A is a simplistic depiction of an autoloading device, used toselectively modify or coat surfaces of differentially coated devices,showing how a solvent, flowing in from the bottom, pushes the modifyingor coating agent up in the chamber, thereby loading the nano-volumechannels.

FIG. 24B is another view of the same autoloading device and chamber,wherein multiple platen are shown held within the autoloading chamber bygrooves in the walls of the autoloading chamber.

FIG. 25 shows an array in accordance with the present invention, whereinone section of the array is enlarged as an inset, lower left, and asingle row of that section of the array is enlarged yet again as anotherinset (lower right) showing a cross-sectional view of a row of channelsor through-holes in the array, with a blocked of loaded channel depictedon the left of the row inset, and five other empty channels, displayedin cross-section, visible to the right of this inset.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

Definitions

The following terms shall have the meanings indicated unless the contextotherwise requires:

“Physicochemical Property” as used herein means any property involvingthe principles of physics and chemistry, alone or in combination,including but not limited to any property of a substance, reaction,molecule, event, process relating to physical state, electronicstructure, electronic principles, thermodynamic principles, atomicstructure, atomic principles, molecular composition and geometry,chemical composition, chemical reactivity, valence state, oxidationstate, oxidation potential, reduction potential, molecular structure,molecular composition, molecular principles, aromaticity, spatialorientation, isomeric form, stereochemical orientation, surface tension,refractive index, wetability, water solubility, density, melting point,boiling point, conductivity, and absorption, luminescence, emission andreflection properties and other light/energy related properties definedby wavelength phenomenon, for example, color and color changes.

Other examples of physicochemical properties include but are not limitedto those relating to hydrophobicity, lipophilicity, hydrophilicity,protein sequence, intramolecular interactions, intermolecularinteractions, two- and three-dimensional structure, for example proteinprimary, secondary and tertiary structure, nucleic acid sequence andstructure, antibody structure, enzyme structure, morphology, propertiesrelating to protein-protein interactions, protein-small moleculeinteractions, nucleic acid-nucleic acid, nucleic acid-protein, ornucleic acid-small molecule interactions, antibody-antigen interactions.The term also encompasses the idea of an intermediate property that iscapable of being altered.

“Channel” as used herein means a space defined by an interior surface orsurfaces connecting at least one exterior surface, wherein the channelis capable of confining a liquid, or in which liquid flows, diffuses, orresides. Channels may be through-holes, troughs, passages, etchedgrooves, contract-printed prescribed alignment patterns, wherein thealignment is aligned or random in space, interconnected with otherchannels or not.

“Through-hole” as used herein means a cylindrical or other shapedchannel open at two ends, so that liquid can flow through. It is oftenreferred to as a microwell, a channel, or micro-volume sample chamber.The volume is such that it may be less than 1 μL, or less than 400 nL,or as little as several nL. Liquid sample is placed therein, and held bysurface tension, aided by the nature of the differential coating on theexternal surface with which the through-hole is in communication. Anano-volume channel, or sample holder, may be similarly sized, byvolume, as a micro-volume channel, through-hole or sample chamber,though typically a nano-volume channel or through-hole has a smallercapacity for a liquid sample than a micro-volume channel/through-hole.

“Trough” as used herein means a particular type of channel having aconcave interior surface defining two continuous sides and a bottom butopen on the third side, the two sides in communication with an exteriorsurface.

“Hydrophilic” as used herein means that which has an affinity for water,of absorbing water, mixing with water, dissolving in water, andinteracting with water such as through hydrogen bonding. Hydrophilicmaterials and compounds may be completely hydrophilic, or have regionsof hydrophilicity, such as a protein, or lipid bi-layer component like afatty acid. Hydrophilic materials tend to have ionic or charged regions,or are polar. A hydrophilic molecule or portion of a molecule is onethat typically is electrically polarized and capable of hydrogenbonding, enabling it to dissolve more readily in water than in oil orother “non-polar” solvents. Hydrophilic molecules can establish hydrogenbonds with water molecules, which are favorable by thermodynamics andmakes these molecules soluble in water.

“Hydrophobic” as used herein means that which has an aversion for water.Hydrophobic compounds and materials tend to be electrically neutral andnonpolar, and thus prefer other neutral and nonpolar solvents ormolecular environments. Hydrophobic is often used interchangeably with“oily” or “lipophilic” or “oleophilic.” Hydrophobic refers to thephysical property of a molecule that is repelled by water. Hydrophobicmolecules in water often cluster together to minimize thermodynamicallyunfavorable interfaces or interactions with the polar water molecules.

“Lipophilic” as used herein means that which has an affinity for lipids.Lipophilic is a particular concept within the broad concept ofhydrophobic—i.e. materials which avoid water—but more particularlyrefers to those materials which have an affinity for compounds commonlyreferred to or known as lipids, such as molecules with a long-chainsaturated or unsaturated hydrocarbons tail and a polar, or chargedoxygen-containing head. More generally, a lipid may be thought of as agreasy hydrophobic compound containing carbon, hydrogen and oxygen.Examples of lipids include steroids; phospholipids, neutral fats andcarotenoids and sphingolipids.

“Blocking Agent” as used herein means a material or substance used tophysically block access of a surface, defined space, channel, region ofa surface or regions of a surface or surfaces, from exposure to achemical composition, reagent or process, energy source or exposure, orenvironment. A blocking agent may be a solid, including a low-meltingpoint solid such as a wax or polymer, or a salt that may or may not beapplied in liquid form and then dried; or the blocking agent may be aliquid, such as an oil, or a grease. Other examples of blocking agentsinclude traditional chemistry blocking agents, wherein a functionalmoiety on a particular chemical composition is reacted with a standardreagent known in the field to create a modified moiety that is “blocked”or protected from reacting with a later reagent in a particular reactionscheme. For both the physical blocking agents and traditional chemistryblocking agents, the blocking agents are removed after protecting theparticular region, or moiety from the chemical reagent of process,energy source or exposure

“Modifying Agent” as used herein means a material or reagent that iscapable of modifying a surface, a composition, a structure, aphysicochemical property or particular characteristic, by eitherchemical means, such as a chemical reaction, or physical means, such asblocking. Modifying agents may be blocking agents, and blocking agentsmay be modifying agents. Both may be coating agents.

“Coating Agent” as used herein means “a material which may be applied toa surface to physically cover all or part of the surface. A coatingagent may be homogenous in its chemical composition and/or chemicalproperties, physical composition and/or physical properties, or it maybe heterogeneous, such as a gradient coating, a multifunctional,multicompositional or multilayered coating. A coating may be appliedchemically, wherein the coating adheres to the coated surface throughchemical bonds, such as covalent bonds, or it may be applied as aphysical coating, wherein the coating adheres to the coated surfacethrough physical interactions such as electrostatic forces, hydrogenbonds, surface tension, Van der Waal's forces, polar interactions anddipole forces, for example.

“Uniform coating” as used herein means a coating, layer, surfacecomposition or deposition covering a surface that is homogeneousrelative to chemical, molecular, or physical state or properties,although the thickness and nature of the coating may vary at any givenlocation on the surface relative to another location. Conversely, anon-uniform coating is a coating, layer, surface composition ordeposition covering a surface that is heterogeneous relative tochemical, molecule or physical state or properties, and may also vary inthickness and surface appearance from one region of the coated surfaceto another.

“Nano-volume specified reaction” as used herein means a particularreaction run in a nanoliter volume scale, such that the total reactionvolume is generally less than a microliter.

“Dissolvable” as used herein means a material, compound, substance oragent whose physical state can be change through heat, hydration,salvation, or other means at will. Thus a dissolvable substance may be awax, which melts to a liquid upon treatment with heat, or a salt orpowder which dissolves upon addition of water or other liquid.

“Physisorbed” as used herein means that the forces holding a substanceto a surface do not involve the making or breaking of chemical bonds,but rely on purely physical interactions for adherence, absorption,affinity or similar interactive phenomena.

In illustrative embodiments, a system and method for registering alocation of a dispenser array relative to a receptacle array ispresented. The receptacle array, which may be a microfluidic array, isrigidly coupled to a frame, while the dispenser array is movable inrelation to the frame. A first camera is in rigid association with oneof the dispenser array and the frame. The first camera is capable ofviewing a fiducial reference in rigid association with the other of thedispenser array and the frame when the dispenser array is in analignment position. Quantities related to a vector displacement from thealignment position to a fixed position on the microfluidic array aredetermined. The quantities thus determined are used to guide positioningof the dispenser array relative to the microfluidic array. Details arediscussed below.

FIG. 3 shows a system 300 for registering a dispenser array 301 to areceptacle array, which may be, without limitation, a microfluidic array303, in accordance with one embodiment of the invention. The dispenserarray 301 typically includes one or more fluid dispensing elements atexpected positions in a plane. The dispenser array 301 may be, withoutlimitation, an array of transfer pins capable of being loaded withtransfer liquid. The microfluidic array 303 may be a platen or chip witha plurality of closed and/or through-hole sample wells at expectedpositions, as described above.

As shown in FIG. 3, the microfluidic array 303 is rigidly coupled to aframe 305, while the dispenser array 301 is movable in relation to theframe 305. Note that alternatively, the dispenser array 301 may berigidly coupled to the frame 305, with the microfluidic array 303movable in relation to the frame 305.

The dispenser array 301 is, without limitation, capable of movingindependently in approximately three mutually orthogonal directions. Forillustrative purposes, the three directions will be referred to as anx-motion axis 307, a y-motion axis 309, and a z-motion axis 311. Invarious embodiments, a gantry with two parallel tracks 313 and 315 maybe attached to the frame 305 to assist in moving the dispenser array301. Due to various tolerances, there is typically skew fromorthogonality between the directions, such as between the x-motion axis307 and the y-motion axis 309.

A controller 325 may be used to control motion of the dispenser array301 relative to the frame 305. Controller 325 may include, withoutlimitation, a robotic motion control system, various circuitry, and/or aCentral Processor Unit (CPU) that may include memory and beappropriately pre-programmed or configured to be loaded with anappropriate program. Memory may include, for example, a diskette, afixed disk, a Compact Disk (CD), Read Only Memory (ROM), ErasableProgrammable Read-Only Memory (EPROM), and/or Random Access Memory(RAM).

The controller 325 may further include one or more encoders thatprecisely determine relative position of the dispenser array 301. Forexample, an encoder for each motion-axis 307, 309, 311 may be provided,such that a set of coordinates identifies the relative position of thedispenser array 301.

The system 300 includes one or more vision means for viewing either orboth the dispenser array 301 and the microfluidic array 303. The visionmeans may include, for example, one or more sensors for detectingposition of either or both the dispenser array 301 and the microfluidicarray 303. The sensor may be, without limitation, an optical or acousticsensor.

In an illustrative embodiment, the vision means includes a first camera317 in rigid association with the dispenser array 301. The dispenserarray 301 may be capable of rotating about a fixed point relative to thefirst camera 317. As shown in more detail in FIG. 4, the dispenser array301 may be mounted to a rotary stage 319 via a dispenser holder 321. Therotary stage 319 permits controlled rotation of the dispenser array 301about a center of origin 401, the center of origin 401 being in rigidassociation with the dispenser array 301. An alternative design may,rotate the microfluidic array 303 instead of, or in combination with,the dispenser array 301.

Referring back to FIG. 1, a second camera 321 may be used, among otherthings, to determine the orientation of the dispenser array 301 relativeto the camera 317. The second camera 321 may be, without limitation,rigidly mounted to the frame 305.

In an embodiment of the invention, a World Coordinate System (WCS) maybe defined whose position is fixed relative to, for example, the frame305, and to which other elements in the system 300 will be referenced.The Origin of the WCS (O_(W)) may be, without limitation, defined by afixed fiducial on the frame 305. Relationships between the WCS, thefield of view of the first camera 317 defined by a First CameraCoordinate System (FCCS), the field of view of the second camera 321defined by a Second Camera Coordinate System (SCCS), the position ofdispenser array 301, and/or the position of the microfluidic array 303are determined. Each of the various coordinate systems may be, withoutlimitation, a Cartesian coordinate system following the right hand rule,however other coordinate systems known in the art may be used.

The x-motion axis 307 of the first camera 317/dispenser array 301 may bedefined to be parallel to the x axis of the WCS, while the y-motion axis309 of the dispenser array 301 is not guaranteed to be parallel to the yaxis of the WCS. The angle between y-motion axis 309 and the y axis ofthe WCS, β, is assumed to be fixed after the gantry is initialized.

FIG. 5 shows an illustrative process of registering the dispenser array301 to the microfluidic array 303, in accordance with one embodiment ofthe invention. The process begins at step 502, in which a spatialrelationship between a location in the field of view of the First Camera317 and a fiducial reference is determined. For exemplary purposes, thefiducial reference will be defined as the origin (O_(W)) of the WCS.

Various associations between the FCCS and the WCS may be determined. TheFCCS may be defined, for example, such that the origin of the FCCS,O_(F), is the center of the first camera's field of view. The x and yaxis of the FCCS makes an angle with respect to the x and y axis of theWCS, which is determined. Furthermore, the pixel resolution of the FirstCamera with respect to the WCS, and a transformation between positionsin the FCCS and the WCS, are determined.

FIG. 6 shows the relative positions of the WCS 601, the FCCS 603 and thedispenser array 605. Typically, the first camera 317 cannot be mountedso that the y-axis 607 of the FCCS perfectly corresponds to the y-motionaxis 309, but rather the y-axis of the FCCS makes an angle θ_(C) to they-motion axis 309. Accordingly, the angle the first camera 317 makeswith respect to the WCS is θ_(C)+β. These two angles must be decoupledbecause β may change when the motion axes 307 and 309 are initialized.

In order to determine angles β and θ, the first camera 317 may movedsuch that a fiducial reference (which may be fixedly positioned, withoutlimitation, to the frame 305) appears in a plurality of distinctpositions in the field of view of the first camera, as shown in FIG. 7in accordance with one embodiment of the invention. More particularly,the fiducial reference may be placed on the right side of the firstcamera's 317 field of view. The position P₁ of the fiducial is recordedin both the WCS and the FCCS. A move along only the x-motion axis 307will then be made such that the fiducial moves to the left side of thefirst camera's 317 field of view. The fiducial will be searched here andthis point P₂ will be saved in both coordinate systems as before. Theangle made by the two points can be determined as follows:φ_(wx)=Atan 2(ΔY _(W) ,ΔX _(W)),  Angle made by point 1 and 2 in theWCS:φ_(cx)=Atan 2(ΔY _(C) ,ΔX _(C)),  Angle made by point 1 and 2 in theCamera:andθ_(C)+β=θ_(CX)=φ_(wx)−φ_(cx).

Note that since a move was made only along the x-motion axis from point1 to 2, φ_(wx) will be 0 degrees, and θ_(C)+β=θ_(CX)=0−φ_(cx).

Next, the first camera 317 will be moved along only the y-motion axis309 in the positive direction from point 3 to point 4. The angles madeby these two point can be determined as follows:φ_(wy)=Atan 2(ΔY _(W) ,ΔX _(W)),  Angle made by point 3 and 4 in theWCS:φ_(cy)=Atan 2(ΔY _(C) ,ΔX _(C)),  Angle made by point 3 and 4 in theCamera:Andθ_(C)+β=θ_(CY)=φ_(wy)−φ_(cy).

In this case, φ_(wy)=90+β, such that θ_(C)+β=θ_(CY)=90+β−φ_(cy), andθ_(C)+β=0−φ_(cx)=90+β−φ_(cy), leading to θ_(C)=90−φ_(cy).

Note that β can be determined from above as β=φ_(cy)−φ_(cx)−90, butanother method, which is typically more precise, may be used todetermine β as follows.

Referring to FIG. 8, a plate with three accurately known points PR1,PR2, and PR3 may be used to determine β, in accordance with anembodiment of the invention. The points PR1, PR2, and PR3 may form, forexample, three corners of a square, the square having sides with knownlength L. The plate may be attached to the frame 305 such that the twosides of the square defined by points PR1 and PR2, and PR1 and PR3 arealigned as close as possible to the WCS y and x axis, respectively. Thetwo end points PR1 and PR3 of the side that is substantially parallel tothe x-axis of the WCS are searched for using the first camera 317. Asthe end points are searched, the angle, θ_(CX), determined earlier willbe used to transform the two end points PR1 and PR3 to the WCS. Thefirst camera 317 will be moved only along the x-motion axis 307. The twopoints PR1 and PR3 are used to determine the angle the square makes inthe WCS, θ_(S). Next, the two end points PR1 and PR2 that aresubstantially parallel to the WCS y axis will be searched for using thefirst camera 317 by moving the first camera 317 only along the y-motionaxis 309. During this search, the camera angle, θ_(C), only will beremoved. The two end points found by the search are (x1, y1) and (x2,y2+dy), where dy is movement along the y-motion axis 309. β can now bedetermined as:

β=90−θ_(S)−φ, where

$\varphi = {{Atan}\; 2{\left( \frac{{y\; 2} + {dy} - {y\; 1}}{{x\; 2} - {x\; 1}} \right).}}$

Once β is known, the location of two fixed points R1 and R2, which maybe defined by two fixed reticles are saved in memory. The first fixedpoint R1 may be arbitrarily defined as the origin of the worldcoordinate system, while the second fixed point R2 is offset from thefirst fixed point R1 in the y direction. The locations of the two fixedpoints R1 and R2 can now be used to recalculate β. The glass plate withthe four reticles is typically very expensive to manufacturer since theposition of the reticles on it must be very precise. The individualreticles are cheaper since each one has only one reticle. The glassplate is used to compute β, and then that information is used toaccurately establish the relationship between the individual reticles.Once Beta is established, the relationship between the two reticles R1and R2 can be accurately determined. By storing the relationship betweenthe reticles R1 and R2, the system can be easily retrained by examiningwhere reticles R1 and R2 are then next time recalibration is required,without using the plate.

As shown in FIG. 9, the angle θ_(R) can be determined by:

$\theta_{R} = {{Atan}\; 2\left( \frac{y_{R\; 2} - y_{R\; 1}}{x_{R\; 2} - x_{R\; 1}} \right)}$where (X_(R1), Y_(R1))=(0,0), and, as determined with the glass plate,

$\varphi = {{Atan}\; 2{\left( \frac{{y\; 2} + {dy} - {y\; 1}}{{x\; 2} - {x\; 1}} \right).}}$β can then be determined using the equation β=θ_(R)−φ.

Once β is known, points in the field of view of the first camera 317 canbe transformed to the WCS using the following equation:

$\begin{bmatrix}x_{W} \\y_{W} \\1\end{bmatrix} = {\begin{bmatrix}{C\;\beta\;\theta_{c}} & {{- S}\;\beta\;\theta_{c}} & {{dx} - {{dyS}\;\beta}} \\{S\;\beta\;\theta_{c}} & {C\;\beta\;\theta_{c}} & {{dyC}\;\beta} \\0 & 0 & 1\end{bmatrix}\begin{bmatrix}x_{FC} \\y_{FC} \\1\end{bmatrix}}$where Cβθ_(C)=Cos(β+θ_(C)), Sβθ_(C)=Sin(β+θ_(C)), Cβ=Cos(β) andSβ=Sin(β).

Referring to FIG. 7, the first camera 317 resolution can now bedetermined by transforming P_(1W) and P_(2W) to the FCCS and taking theratio of change in motion in the WCS x direction to the change in thefirst camera's 317 field of view in the x direction.

To transform P_(1W) and P_(2W) to the first camera's 317 frame ofreference P_(1WC) and P_(2WC):

$\begin{pmatrix}{Xwc} & {Ywc}\end{pmatrix} = {\begin{bmatrix}{{Cos}\left( {\beta + \theta_{c}} \right)} & {{Sin}\left( {\beta + \theta_{c}} \right)} \\{- {{Sin}\left( {\beta + \theta_{c}} \right)}} & {{Cos}\left( {\beta + \theta_{c}} \right)}\end{bmatrix}\begin{bmatrix}{Xw} \\{Yw}\end{bmatrix}}$for both points

The first camera

${X\mspace{14mu}{resolution}} = \frac{\Delta\;{Xwc}}{\Delta\;{Xc}}$(units per pixel), where ΔX_(WC)=(X_(2WC)−X_(1WC)) and ΔX_(C)=(X₂−X₁).Similarly, the Y resolution of the first camera can be determined by P3and P4.

Referring back to FIG. 5, the spatial relationship between locations inthe second camera's field of view 321 and the fiducial reference issimilarly determined in step 502. More specifically, the SCCS may bedefined, for example, such that the origin of the SCCS, O_(S), is thecenter of the second camera's 321 field of view. The x and y axis of theSCCS makes an angle θ_(2C) with respect to the x and y axis of the WCS,which is determined. Furthermore, the pixel resolution of the SecondCamera with respect to the WCS, and a transformation between positionsin the FCCS and the WCS, are determined. The second camera 321 is fixedin the WCS and therefore the location of the second camera 321 is alsodetermined.

The second camera's 321 angle θ_(2C) is determined by following aprocedure similar to that used to determine θ_(C) above. The differenceis that a dispenser on the dispenser array 301 will be used as afiducial reference, with the dispenser being move relative to the secondcamera 321 along the x-motion axis 307.

Once the camera angle θ_(2C) is known, the pixel resolution of thesecond camera 321 can be determined following a similar procedure as forthe first camera 317.

Finally, the location of the second camera's 321 (e.g., the center oforigin in the SCCS) is determined in the WCS by placing a fiducialreference in the second camera's 321 field of view and locating it withboth the first camera 317 and the second camera 321. Using the firstcamera 317, the fiducial reference's position in the WCS can bedetermined, allowing a position in the field of view of the secondcamera 321 to be determined in the WCS.

Referring back again to FIG. 5, the location of the dispenser array 301relative to the fiducial reference is determined in step 506. Thedispenser array 301 may be mounted in rigid association with the firstcamera 317, and in various embodiments may be rotated about a center ofrotation 401 (see FIG. 4). The center of rotation 401 can be determinedby training a single dispenser, for example, a single pin, of thedispenser array 301 in the field of view of the second camera 321. Thedispenser array 301 is rotated such that the trained pin remains in thefield of view of the first camera 317. By rotating the pin to at least 3different positions within the field of view of the second camera 321, afit circle algorithm can be used to determine the center of rotation 401in the WCS. This may be done with several pins, with the results beingaveraged. As shown in FIG. 10, a vector T_(PC) is formed between thefirst camera's 317 O_(C) and the center of rotation O_(P) of thedispenser array 301, in accordance with an embodiment of the invention.The vector T_(PC) can consequently be determined by vector subtraction.

Upon determining the center of rotation O_(P) of the dispenser array301, the center of the dispenser array 301 and the positions of thedispensers on the dispenser array 301 relative to the O_(C) aredetermined, along with the angle that the dispenser array 301 makes withthe WCS. The angle will be used to determine an offset to the dispenserarray 301 rotary axis when aligning the dispenser array 301 to themicrofluidic array 303.

FIG. 11 is a layout of an exemplary dispenser array 1101, in accordancewith one embodiment of the invention. The dispenser array 1101 includes48 pins 1107 positioned on a grid defined by a grid x-axis 1103, and agrid y-axis 1105 orthogonal to the grid x-axis 1103. The pins 1107 forma 4 pin (along the x-axis 1103) by 12 pin (along the y-axis 1105) array.The angle that the dispenser array 301 makes with the WCS may bedefined, for example, as the angle between the grid x-axis 1103 of thedispenser array 301 and the WCS x-axis.

In various embodiments, a best fit algorithm may be utilized todetermine the angle the dispenser array 301 makes with the WCS. Ofcourse, other algorithms known in the art may also be used. The best fitgrid is determined by best fitting lines in the grid x an y axisdirections 1103 and 1105. For example, with regard to the dispenserarray of FIG. 11, the pins may be sorted in four groups of twelve (alongthe grid y-axis 1105) and twelve groups of four (along the grid x-axis1103). A best fit line is applied to each group. The average slope andintercept of the four lines in the grid y-axis 1105 direction and theaverage slope and intercept of the twelve lines in the grid x-axis 1103direction is used to determine the center of the grid 1110. The centerof the grid 1110 is the intersection of the two average lines. The angleof the grid relative to the WCS can be determined by taking the averageangle of the vertical and horizontal lines. Ninety degrees is subtractedfrom all the vertical lines. The vertical lines may be weighted threetimes more than the horizontal lines since they have as many times morepoints.

Worst pin positions may be discarded from the best fit algorithm, so asto provide a better fit for the majority of pin positions. The algorithmmay be applied a predetermined amount of times, with earlier resultsdetermining the pin positions that are farthest from the line. Thealgorithm is typically run again after removing the bad pin positions.The number of pin positions in each line may determine how many pinpositions are discarded. For example, only one point may be discarded ineach horizontal line (i.e., lines parallel to the grid x-axis 1103),with three points being discarded in each vertical line (i.e. linesparallel to the grid y-axis 1105). Furthermore, bad pins can now beidentified or checked against known specifications. In variousembodiments, a template of expected pin locations may be aligned withone or more viewed pin positions to aid in the alignment process.

Referring back to FIG. 5, the location of the microfluidic array 303relative to the fiducial reference is determined, step 508. Themicrofluidic array 303 is placed in the field of view of the firstcamera 317 such that the four corners of the microfluidic array 303 canbe examined. The location, orientation, and the stretch of themicrofluidic array 303 in the WCS is determined. The microfluidic array303 may be in the form of one or more chips, as discussed above, thatare arranged on one or more trays. For example, each chip may includesample wells that are grouped into a 4 by 12 array of 5-well by 5-wellsub-arrays In another embodiment, the sub-arrays may be 8-wells by8-wells or any other convenient number. For alignment, only the lowerleft hole corresponding to the pins on each of the four corner arrays isused. Other embodiments may of course use other holes for aligning. Forexample, the extreme corner holes on each of the four corner arrays maybe used. A controller is capable of navigating the first camera 317 toeach chip on the tray and aligning (i.e. determining the WWC of eachchip), given the row and column of the desired chip on the tray. Thebase location of each of the trays is stored in the registry.

The base location may be defined by the lower left corner of the chip303 located in the upper right corner of the tray. The registry alsostores the pitch of the rows and columns of each chip in the tray. Invarious embodiments, there are two rows and six columns on each tray,with the pitch between the columns 1-2, 3-4, and 5-6 is different thanthe pitch between columns 2-3 and 4-5. Therefore, there are twodifferent pitch values stored ‘Pitch1’ for the first set and ‘Pitch2’for the second set. The pitch of the holes in the sub-grid and the pitchof the pins is also known. Using all this information, the system cannavigate itself to each of the four corners of any given chip and alignit.

For example, referring back to FIG. 2, the first camera 317 will move tothe lower left corner and locate the lower left hole 21 of the sub-grid.The first camera 317 will then move to the lower right corner and locatethe lower left hole 22 for the sub grid. This is repeated for the lowerleft hole 23 of the upper right corner sub grid and finally the lowerleft hole 24 of the upper left corner sub grid.

As shown in FIG. 12, the four points 1201-1204 (found in this example atthe extreme corners of the chip) are used to calculate the angle of themicrofluidic array 303 in the WCS. If the chip is aligned at a desiredangle, the diagonals will form Angle1 and Angle2 with an arbitraryhorizontal line. But if the chip is rotated as shown in FIG. 12(b), theangles formed by the diagonals and the arbitrary horizontal line will beslightly different. The difference between the angles will be used tocalculate the angle of the microfluidic array 303 in the WCS. Inparticular, rotation1=Angle1′−Angle1 and rotation2=Angle2′−Angle2.Ideally, rotation1 would equal rotation2. In practice they will beslightly different. Rotation1 and rotation2 may then be, for example,averaged, to determine the angle of the microfluidic array 303 in theWCS.

The center 1208 of the microfluidic array 303 is determined bycalculating the center of the found rectangle. The stretch in xdirection is calculated by comparing the horizontal sides of the foundrectangle and the theoretical rectangle. The stretch in y is calculatedby comparing the vertical segments. If the stretch in either directionis greater then a predetermined value, the alignment for themicrofluidic array 303 fails.

If the stretch is within limits, the microfluidic array 303 is thenregistered with the dispenser array 301, step 510 of FIG. 5. Thelocations and orientations of the dispenser array 301 and themicrofluidic array 303 relative to the fiducial (e.g., O_(W) of the WCS)have been determined, as described above. A vector displacement betweenthe center of the microfluidic array 303 and the center of dispenserarray 301 can thus be determined, and the first camera 317/dispenserarray 301 moved accordingly. Furthermore, the dispenser array 301 isrotated using rotary stage 319, such that the angle of the microfluidicarray 303 matches the angle of the dispenser array 301.

In accordance with other embodiments of the invention, a method ofregistering a location of a dispenser array in relation to a receptaclearray is provided. FIG. 13 shows a system 1300 wherein one dispenserelement 1301 can be positioned to dispense fluid into, for example, anopposing through-hole in a microfluidic array 1302. A coordinate systemexternal to the microfluidic array 1302 is defined by a rigid frame towhich the dispenser element 1301 is fixed. Two or more cameras 1305 arepositioned on the opposing side of the microfluidic array 1302 from thedispenser element 1301 under specified through-holes of the microfluidicarray 1302. The dispensing element 1301 could be a pin, as describedpreviously. The pin 1301 is actuated to move in a rectilinear coordinatesystem (XYZ) 1310. The microfluidic array 1302 is rigidly fixed in theXY plane defined by the motion of the pin and the array is actuated torotate in the XY plane with the axis of rotation (Θ) parallel to the pin(Z axis).

Important elements within the system 1300 include: (i) relativecenter-to-center positional accuracy of the through-holes; and (ii)accurate measurement of pin 1301 and microarray 1302 XYZΘ displacement.The precision and accuracy of the center-to-center spacing ofthrough-holes in the microarray 1302 is such that selection of at leastthree through-holes as alignment fiduciaries defines a XY coordinatesystem locating all the through-holes in the microarray 1302. The pintranslation and microfluidic array rotation actuators typically haveseparate means to measure the displacement (linear or angular). This maybe accomplished be by counting steps in a pre-calibrated stepping motordrive or with a separate displacement sensor. The center-to-centerspacing of the pins 1301 (in embodiments having a plurality of pins) inthe pin array may be advantageously an integral of the through-holecenter-to-center spacing, with the error in the spacing being typicallyless than the precision of the distance between the array through-holes(<+−25 micrometers) by at least a factor of 5.

The Z axis coordinate of the tip of the pin 1301 relative to thethrough-hole array 1302 surface is determined by recording the distancethe pin 1301 is moved to bring it into contact with the through-holearray 1302 surface.

One or more cameras 1325 are positioned opposite each fiducialthrough-hole 1320. The cameras are typically rigidly mounted and are notmoved relative to the microfluidic array 1302 nor pin 1301.

In various embodiments, determination of the XY coordinate system may beaccomplished as follows:

A minimum of three through-holes are selected, one in each corner of themicrofluidic array 1302, as fiduciaries 1320 for coordinate systemdetermination.

The pin 1301 is positioned above a fiducial through-hole 1320 and acamera 1325 is positioned below.

The pin 1302 is moved in XY until the image of the pin tip is positionedin the center of the fiducial through-hole 1320.

The pin 1302 is moved to the second and third fiducial through-holes1302 and coordinate vectors of the pin center relative to thethrough-hole center is measured.

A coordinate transformation is then enacted which first rotates themicrofluidic array 1302 to align the through-hole array axes to the pin1301 translation axes (see, for example, FIG. 14). The pin 1301 thenre-visits the first through-hole 1320, positioned relative to thethrough-hole center and then moved to determine the lateral offset fromthe other two fiducial through-holes 1320. The angular offset isrecomputed, the microarray 1302 is rotated and the positioning procedurerepeated until the position of the pin tip relative to each fiducialthrough-hole center is less than +−5 micrometers.

The Z axis is determined by starting from a Z position above themicrofluidic array 1302 surface. After the XY position of the pin 1301is determined, the pin 1301 is brought into contact with the fiducialthrough-hole 1320 to determine the distance from the inserted pinposition and a starting point above the microfluidic array 1302 surface.

In various embodiments, a coherent fiber bundle may illuminate andtransfer an image of the through-hole to project onto a camera. Onelight source may be used for multiple fiber bundles, with one cameraassociated with each imaging fiber bundle.

One camera can be multiplexed to image two or more spatial positions onthe microfluidic array 1302 by the following method. A coherent fiberbundle transmits an image of a fiducial through-hole for projection ontoa sub-segment of the camera. At least three, if not four, through-holeimages can be multiplexed onto a common camera image with this approach.

FIG. 15 shows a system 1500 wherein the dispenser array 1550 containsmore than one dispenser element 1501, with the number of pins 1501 equalto the number of subarrays in a microfluidic array 1502. Each pin isregistered to a through-hole in the same relative position in eachsubarray with this method. In alternative embodiments, the dispenserarray may have a pin for each throughhole, with the center-to-centerspacing of the dispenser elements substantially matching the pitch ofthe through-hole array.

A through-hole array can be used as an alignment mask to pre-positionthe pins relative to the through-hole array. Used as a mask, the holesin the mask through-hole array are sized to accept the pins in a slidingfit. Each pin is free to move in the Z axis yet constrained relative tolateral (XY) translations and rotation (Θ).

Detailed Examples of Preferred Embodiment

Masking/Blocking/Protecting of Some or all Holes or Etched Areas Priorto Deposition:

Methods are disclosed for using force-loading to fill holes with amaterial that prevents deposition of a surface-modifying agent, whileallowing the exterior surface to be coated. For example, large amountsof water can prevent the deposition of silane-based coatings because thesilanes react with the water prior to reaching the surface.

One technique is to force-load water into holes or etched areas throughthe use of liquid exchange with ethanol (a lower surface energy fluid)and then expose the OA or etched plate to a silane vapor. It is alsopossible to force-load the etched areas or holes through the use ofvacuum or momentum. For example, holding a hydrophobically coated OAplate under running water can force water into the holes while runningoff of the exterior surfaces; or a flowing gas or fluid can be used toprevent deposition of a surface-modifying agent in the holes of an OAplate, while allowing modification of one side of the OA.

For example, by arranging the flow of nitrogen such that the gas isforced to flow through the holes in an OA plate (from “back” to “front”)while the plate is in a vapor deposition chamber, and preventing throughthe use of o-rings or other gasketing materials the exposure of the“back” side of the OA plate while the “front” is exposed to the vapor ofa surface-modifying agent such as fluoro-trichlorosilane, the interiorsurfaces of the holes are blocked from modification. After coating the“front” side of the OA plate, the plate can be reversed such that the“front” and “back” designations are interchanged. Then, the process canbe repeated resulting in an OA plate with modified exterior surfaces andun-modified interior surfaces.

A solid material can be used to block holes or etched areas duringsurface-modification of the rest of the OA. The material is chosen tohave a melting point that allows it to remain solid during alldeposition and modification steps, but to be melted away from allprotected surfaces after deposition at a temperature that will notadversely affect the substrate material or any coatings applied thereto.Alternately, a solid or semi-solid material can be chosen that can bedissolved by a solvent that will not affect the substrate or coatingsapplied thereto.

For example a waxy material like polyethyleneglycol (PEG) can be easilyselected that has the required melting temperature ranges forlow-temperature deposition processes, since PEG is available in a largevariety of molecular weights giving it a large range of melting points.The PEG or other such material can be solidified in the OA holes oretched areas of substrates after flowing a liquid phase of the materialinto necessary areas. Reusable masks can be used to prevent exposure ofthe exterior surfaces.

One method is to stack a large number of OA plates together such thatall through-holes align and the group is tightly held together.Submerging in a liquid phase of a blocking material then fills all ofthe holes continuously while exposing only the exterior surfaces of theOA plates on each end of the stack (“masks”) and the outside edges ofeach of OA plate which are unimportant. Then the stack can bedisassembled and the OA plates subsequently coated. After coating, theplates are heated and the blocking material melts and is removed.

Another method is to coat the entire OA plate, including the holes, witha PEG-type material or a low melting point metal (e.g. gallium and itsalloys). Then, the exterior surfaces are sanded, scraped, lapped,polished, ground or otherwise mechanically treated to remove theblocking material from those areas and the OA plates subsequentlycoated. After coating, the plates are heated and the blocking materialmelts and is removed.

Depending on the reaction rates, the deposition process could beperformed at low temperature (at or below room temperature) where thematerial blocking the channels is solid. Raising the temperaturepost-deposition will cause the blocking material to become liquid forremoval from the channel.

Masking/Blocking/Protecting of Some or all of the Surface Prior toDeposition:

In some embodiments, a mask can be used to prevent the surface-modifyingagent from affecting the exterior surface during deposition and coatingof the OA holes or etched areas. For example, a mask is used duringmanufacture of the OA plates themselves. By not stripping this maskduring manufacture of the OA plate, the mask will still be bonded to theOA plate and provide protection of the exterior surfaces duringsurface-modification of the etched areas or OA holes.

Alternatively, on OA plate can be used to block exterior surfaces ofother OA plates by simply placing it atop the other. 2 OA plates couldbe used to make a “sandwich”, with a target plate between two otherplates used as masks. The exterior plates could be reused many times.

Examples of the usefulness of blocking include:masking/blocking/protecting of some or all holes or etched areas afteruniform deposition and prior to etching/dissolving/modifying/destroyingsurface coating; masking/blocking/protecting of some or all of thesurface after uniform deposition and prior toetching/dissolving/modifying/destroying holes' surface coating.

Another embodiment discloses the use of force-loading to fill holes witha material that contains a surface-modifying agent, while allowing theexterior surface to be unchanged. One technique is to force-load liquidcontaining a surface-modifying agent into holes or etched areas throughthe use of liquid exchange with ethanol (a lower surface energy fluid).It is also possible to force-load the etched areas or holes through theuse of vacuum or momentum. For example, holding an OA plate in a streamof liquid containing surface-modifying agents allows the holes to beloaded while keeping the exterior surfaces dry.

In other embodiments, the surface-modifying agents can be echants (suchas a base to remove any present coating), oxidants (to modify thepresent coating), or other chemicals that allow addition to ormodification of the surface chemistry.

Moreover, an OA plate can be used to block exterior surfaces of other OAplates by simply placing it atop the other. 2 OA plates could be used tomake a “sandwich”, with a target plate between two other plates used asmasks. The exterior plates could be reused many times.

For example, a masked OA could be exposed to uv-radiation of wavelengthless than 200 nm in the presence of ozone. Systems are available fromUshio America, Inc. that generates uv of 172 nm wavelength. The actionof the energetic photons and the oxygen singlet and triplet moleculescan destroy most organic bonds allowing removal of the surface coatingin the interior of the OA holes or the etched areas of a substrate.

Masked UV Initiated In Situ Polymerization to Selectively Coat thePlanar Non-Etched and Through Hole Surfaces Using a Photo-Reactive Film

The planar non-etched and etched areas of the open array can beuniformly coated with a photo-reactive material by treating the surfaceswith a vinyl-terminated silane, for example, then force load the throughholes with a PEGylated monomer such as Polyethylene glycol 400monoethylether monomethacrylate (PEGMA), for example, to grow a PEGlayer by in-situ polymerization under UV light at 360 nm of wavelengthin the presence of a photo initiator such as MEN or TENTED and using aphoto-mask such as a second OA, to protect the planar surface from UVradiation. Alternatively, the polymerization could be carried out usingan appropriate thermo-initiator.

After the internal surface of the open array is polymerized andblocked-terminated, the planar non-etched surface can be selectivelyreacted with a hydrophobic monomer such as 10—heptadecafluorodecylacrylate by in-situ polymerization by immersion in a solution containingthe monomer and appropriate photo or thermo-initiator.

Coating of the Planar Non-Etched Surface of the Open Array by ContactPrinting or Stamping

Stamping or contact printing is another approach for differentialcoatings of the planar and through-hole surfaces in an open array. Inthis approach, a series of stamping and solution-phase modifications areapplied to achieve differential coatings. First, a polydimethylsiloxanepad PDMS is pretreated with a hydrophobic silane derivative such asoctadecyltriclorosilane OTS, for example, and brought into contact withboth sides of the OA. This step coats exterior surfaces of through-holeswith a hydrophobic film. Then, the whole chip is exposed to multiplesolution-phase reactions, where interior surfaces of through-holes arefirst modified with a functional silane film such as vinyl, aldehyde, orepoxy terminated derivatives, and further modified to attach apolyethylene glycol (PEG) film or any other molecule of interest such asproteins, peptides or oligonucleotides. The key characteristics of thisapproach is that once the exterior surface is coated with a hydrophobicfilm by stamping, the film is not affected by a series of chemicalreactions downstream targeted for modifying interior surfaces.

Non-limiting examples of a patterned substrate may be stainless steelplate having etched grooves or patterns, or a stainless steel platehaving through-holes such as a microfluidic sample array (a chip). Inother embodiments, the patterned substrate may be of silicon, polymericmaterials, glass or any other substrate known to those of skill in theart. Various methods of differentially treating the patterned substrateor through-hole array plate may include a series of treatments andreactions. The series of treatments in various embodiments in accordancewith the presently claimed invention may involve inspection; labelingfor tracking during processing; cleaning; coating of the planarnon-etched and/or through-hole substrate surface with a first reagent;treating the through-hole and/or non-etched surface with a reagent thatactivates or modifies the surface for later treatment by producingreactive groups on the etched or through-hole surfaces; treating theetched or through-hole surfaces with a reagent different from that usedto treat the planar non-etched or non-holed surface of the substrate;additional treatment of the etched or through-hole surface to preventreaction with reagents which may be used in later treatments; coatingthe planar non-etched or non-holed surface of the substrate with asecond reagent that either adsorbs or chemically reacts with the firstreagent on the non-etched or non-holed surface; and quality controltests.

A particular embodiment may first uniformly coat the non-etched ornon-holed (and the through-hole) surface by treating with a functionaland hydrophobic silane derivative such as vinyl-terminated silane, forexample, then selectively oxidize the etched or through-hole surfaces byfirst (force) loading with a low surface tension-aqueous phase misciblesolvent such as ethanol followed by loading by mixing of a oxidant suchas a permanganate solution, for example, of 5 mM KMnO₄ and 19.5 mM NaIO₄in deionized water. Other oxidizing agents may include but are notlimited to dichromates and peroxides. This type of chemical modificationis done by incubation in a nonreactive oil or liquid such as aperfluorinated alkane solution for 2 hours, then PEGylation (coveringwith polyethylene glycol or molecules bearing PEG moieties) of theselectively oxidized etched or through-hole surface is done followed byre-loading of the etched or through-holed surface with additionalprotecting PEG, with a final coating of the non-etched or non-holedsurface accomplished by treatment with perfluorosilane using vapor-phasedeposition.

Alternatively, the initial coating of the non-etched or non-holed planarsurface (it should include the internal surface) of the substrate may bedone using liquid phase vinyl deposition with 7-octenyltrichlorosilaneor 10-undecenyltrichlorosilane, for example. The PEGylation mayalternatively be performed using various PEG-silane derivatives, such aslinear, branched or dendritic PEGS. Some examples include methoxy-PEG ofvarious MW, bifunctional PEG derivatives, and star-PEG. Also, aldehyde,epoxy, and carboxylic silane derivatives may be used as an alternativefirst coating of the non-etched and/or etched surface of the substrate.This allows PEGylation of the etched or through-hole surface withoutselective oxidation by controlling parameters such as pH and reagentconcentration. Other PEGylation methods include the use of longer PEGmolecules, such as methoxy-PEG-amine MW 5000 in place of silane-PEGcoatings of lower molecular weights. In addition, the PEG within thePEGylated etched or through-hole surfaces may be cross-linked usinghyperbranched PEG and PEI molecules and appropriate cross-linkermolecules.

In such embodiments, the etched or through-hole surface is treated witha reagent to expose functional group A, followed by exposure to asolution containing a PEG having a terminal functional group B that isreactive with functional group A. The solution may also contain PEGhaving a terminal functional group A, and may contain a catalyst oractivator that facilitates reaction between functional groups A and B toform a covalent bond—i.e., cross-links.

For example, the etched or through-hole surface may be treated to exposea carboxylic acid moiety. A solution is then prepared with, for example,a 6-arm PEG terminated with —NH₂ and a PEG terminated with —COOH, andmay also contain EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride). The carboxylic acid-containing etched or through-holesurface is then exposed to the solution of PEGs and catalyst, and ifnecessary, the solution may be dried on the etched or through-holesurface and incubated at high temperature to improve the yield ofcross-linking between the amine and carboxylic acid groups.

Other embodiments may uniformly coat the non-etched non-holed planarsurface with a film exposing an epoxide group. After treatment withammonia gas to open the ring and expose hydroxyl and amine groups, asolution of PEG bearing amine-reactive and/or hydroxyl-reactivefunctional groups is force-loaded into the etched or through-holesurfaces and incubated as described above, followed by standard batchcoating of the non-etched or non-holed planar surface with a secondreagent to achieve differential coating of the final substrate.

PEGylated etched and through-hole surfaces created neutral hydrophilicsurfaces. Alternatively, specific bio-reactive surfaces may be createdby treating the etched or through-hole surfaces with reagents such asbiotin-streptavidin reagents, antibodies, proteins, nucleic acid probes,small molecules, or promoters of cell-adhesion to create surfacescapable or reacting with specific antibodies, molecules or cells ofinterest.

As can be seen in the embodiment depicted in FIG. 16, a vaporizedcoating material such as a silane 1640 flows from the top and coats theupper external surface 1610 of the substrate 1600. From below, an inertgas 1650 such as N₂ flows through the channels 1620 to protect them fromthe silane, thus differentially coat a device in accordance with anembodiment of the invention, wherein uni-directional flowingfluorosilane gas is used to coat an exterior surface of the device, andopposite-flowing N₂ is used to protect the channels.

A similar embodiment is seen in FIG. 17, wherein bi-directional flowingsilane gas 1600 is used to coat two exterior surfaces 1710 and 1720 ofthe device, and the channels are protected from the silane coating agentby a wax 1700, which may be dissolvably removed later by melting orheating.

FIG. 18 shows another a method for differentially coating a device inaccordance with the present invention, wherein the entire device 1800,including the plurality of nano-volume channels, is first coated with ahydrophobic film 1850—fluorosilane in this case—followed by blocking ormasking of one external surface 1860, such that treatment with UV light1870 strips the silane from the channels but not the masked/blockedexternal surface 1860.

FIG. 19 shows a schematic of yet another method for differentiallycoating a device in accordance with the present invention, wherein theentire device, including the nano-volume channels, is first coated witha hydrophobic film 1900, such as fluorosilane, as depicted here. Thechannels 1920 are then subjected to an etching/modifying agent 1950 suchas strong base, which selectively removes the hydrophobic film fromnano-volume channels 1920.

FIG. 20 depicts yet another way to differentially coat a device inaccordance with the present invention, wherein the device is completelycoated with a reactive film 2000, in this case a reactive silane, afterwhich first a hydrophilic 2010 and then second a hydrophobic film 2020are applied on the channel and external surfaces, followed by selectiveremoval of the hydrophilic and second hydrophobic layers by subjectingthe device, particularly the nano-volume channels, to 360-nm light 2030which catalyzes the release of the hydrophilic and subsequenthydrophobic layers from the surface of the channels, leaving only theoriginal hydrophilic layer 2010 in the channels, and the other layers2020 on the external surfaces.

FIG. 21 shows a method for differentially coating a device using contactprinting or stamping methods in accordance with embodiments of theinvention, wherein a printing pad or stamp pad 2100 is coated with afirst hydrophobic agent 2120 namely polydimethylsiloxane (PDMS), thenlayered with octadecyltriclorosilane (OTS) 2150. By contacting thedevice with the OTS-layered printing pad, at least one external surface2130 of the device is coated with the OTS, thus allowing controlledapplication of compounds to the external surfaces of the device, such asOTS in from the printing pad to the device, after which the internalsurfaces 2140 of the array device may be modified using reagents such asaldehyde-terminated silane and amine-terminated polyethylene glycol(PEG); and carboxyl-terminated PEG+amine-terminated PEG.

FIG. 22 shows the use of forced loading to load channels in ahydrophobically coated device 2200 with ethanol 2210, a lower energy(surface tension) solvent to force load the channels 2220 followed byimmersion of the ethanol-loaded device with an aqueous solution 2230,the lower surface tension ethanol “pulling” the higher energy (surfacetension) water into the nano-volume channels 2140, after which thedevice may be dipped in an aqueous solution 2250 to exchange water foraqueous reagents yielding a hydrophobically coated substrate loaded withhydrophilic reagent 2260, thereby enabling chemical reactions to beconducted in the nano-volume channels as the device is incubated in oil,for example

FIGS. 23A and 23B show a process for differentially coating the surfacesof a patterned substrate in accordance with an embodiment of thepresently claimed invention. in FIG. 23A, the substrate 2300 isoriginally treated so that all exposed surfaces are activated with anactive compound 2310 after which the all substrate surfaces are modifiedwith an active hydrophobic component 2320.

In FIG. 23B additional steps of the process can be seen, wherein anoxidizing agent selectively oxidizes the interior surface 2330 of thesubstrate after which the inner surface is selectively modified with ahydrophilic agent 2340. At this point, the inner surface is coated withadditional agent hydrophilic agent 2350 and dried.

FIG. 23C shows the final steps of the process wherein the selectivelycoated interior surface 2350 is coated with a hydrophobic coating agent2360 after which the sustrate 2300 is rinsed with the hydrophobiccoating agent, selectively rinsing off from the interior surface allcoatings not chemically bound leaving only bound hydrophilic agent 2370.

FIG. 24A is a simplistic depiction of an autoloading device 2400, usedto selectively modify or coat surfaces of differentially coated devices2420, showing how a solvent 2430, flowing in from the bottom, pushes themodifying or coating agent 2440 up in the chamber, thereby loading thenano-volume channels.

FIG. 24B is another view of the same autoloading device and chamber,wherein multiple platen 2420 are shown held within the autoloadingchamber by grooves 2460 in the walls of the autoloading chamber 2400.

FIG. 25 shows an array 2500 in accordance with the present invention,wherein one section of the array is enlarged as an inset 2510, lowerleft, and a single row of that section of the array is enlarged yetagain as another inset 2520 (lower right) showing a cross-sectional viewof a row 2540 of channels or through-holes 2550 in the array, with ablocked or loaded channel 2560 depicted on the left of the row inset,and five other empty channels 2570, displayed in cross-section, visibleto the right of this inset.

In various embodiments, the disclosed system and method may beimplemented as a computer program product for use with a computersystem. Such implementation may include a series of computerinstructions fixed either on a tangible medium, such as a computerreadable media (e.g., a diskette, CD-ROM, ROM, or fixed disk) ortransmittable to a computer system, via a modem or other interfacedevice, such as a communications adapter connected to a network over amedium. Medium may be either a tangible medium (e.g., optical or analogcommunications lines) or a medium implemented with wireless techniques(e.g., microwave, infrared or other transmission techniques). The seriesof computer instructions embodies all or part of the functionalitypreviously described herein with respect to the system. Those skilled inthe art should appreciate that such computer instructions can be writtenin a number of programming languages for use with many computerarchitectures or operating systems. Furthermore, such instructions maybe stored in any memory device, such as semiconductor, magnetic, opticalor other memory devices, and may be transmitted using any communicationstechnology, such as optical, infrared, microwave, or other transmissiontechnologies. It is expected that such a computer program product may bedistributed as a removable media with accompanying printed or electronicdocumentation (e.g., shrink wrapped software), preloaded with a computersystem (e.g., on system ROM or fixed disk), or distributed from a serveror electronic bulletin board over the network (e.g., the Internet orWorld Wide Web).

Although various exemplary embodiments of the invention have beendisclosed, it should be apparent to those skilled in the art thatvarious changes and modifications can be made which will achieve some ofthe advantages of the invention without departing from the true scope ofthe invention. These and other obvious modifications are intended to becovered by the appended claims.

In order to produce an OpenArray™ Chip for performing biological assays,a chip substrate, or a plate with holes, must be differentially coated.The current design of the chip is slightly smaller than a microscopeslide in outer dimensions, but is only 300 μm thick. It has 3072 vias orholes in it, each 300 μm in diameter on a 500-μm pitch. In addition, thecoating process must be applicable to other size chips, such as our25,000-hole chip, as well as higher density (smaller hole) chips.

The exterior surfaces of the chip substrate is preferably hydrophobic,while the interior surfaces of the through-holes is preferablyhydrophilic and compatible with a specific assay run insidethrough-holes. Hydrophobicity of exteriors prevents any crosstalkbetween the filled through-holes by maintaining a solution within theholes, and also inhibits any adsorption of organics on the surface ofthe chip during sample introduction. The hydrophilic coating ofinteriors of the holes enables instantaneous and spontaneous loading of˜30 nL volume into the holes upon loading. In addition, the interiorcoating needs to be biocompatible as well as hydrophilic becauseminiaturized biological assays run in the holes can be affected by thesurface in such high surface-to-volume environment. In otherapplications, the chemistry of the interior of the wells must beflexible so that the surface of the holes can be functionalized to bindmolecules of interest to the surfaces.

Substrates

Originally, the feasibility of this technology was demonstrated withsilicon and glass as substrates. Both materials can be processed usingphotolithographic techniques resulting in fine control of features suchas the holes needed for Thru-Holerm technology. Also, the coatingchemistry necessary for performing biological assays in glass iswell-known, and these chemistries are applicable to silicon as well.Both silicon and glass substrate chips can be coated satisfactorily forbiological assays. However, there are problems with both.

Stainless steel is an attractive material for disposablemicrofluidic-based chips thanks to low material cost and readilyavailable inexpensive fabrication processes. One of the major challengesin employing stainless steel for producing a microfluidic chip forbiological applications is the control of its surface properties.Through surface modification with molecular-level thin films, stainlesssteel chips with differential hydrophilic and hydrophobic surfaces andspecific biologically active surfaces are produced, as desired.

In developing a coating process for stainless steel chips, two majorhurdles were overcome: understanding and control of steel surfaceproperties and differential coatings for exteriors and interiors ofthrough-holes. The coating processes disclosed herein provide solutionsto such challenges and provide steel chips of consistent and reliablequality of coating. In addition, the process is highly compatible withscale-up in manufacturing and the production throughput can be readilyincreased with minimum amount of instrumentation and additional labor.

Coating Background

Many methods for applying differential coatings to chip substrates havebeen attempted. Originally, the methods were variations of a vapor phasedeposition of perfluorinated hydrocarbon silanes. Some of these methodswere vapor phase deposition of the hydrophobic silane with:

-   -   The holes protected by flowing gas through the holes and keeping        the silane from entering the holes    -   The holes protected by a waxy substance that was later dissolved        away.    -   The holes coated with silane but subsequently stripped by using        a mask and high energy uv light in the holes    -   The holes coated with silane but subsequently stripped by using        dip loaded chemistry

Batch coating processes for a stainless steel chip employ novelcombinations of surface modification and microfluidic phenomena. Theprocess is composed of a series of vapor- and solution-phase surfacereactions. Notably, some of the solution-phase reactions in the processare localized within through-holes by taking advantage of microfluidicphenomena, which enables differential coating.

Differential Coating Using Stamping Methods

Stamping works well for differential coatings of exteriors and interiorsof through-holes in a chip. In stamping, a series of stamping andsolution-phase modifications were applied to achieve differentialcoatings. First, a polydimethylsiloxane (PDMS) pad was pretreated withhydrophobic silane such as octadecyltriclorosilane (OTS) and broughtinto contact with both sides of a chip. This step coats exteriorsurfaces of through-holes with a hydrophobic silane film. Then, a wholechip is exposed to multiple solution-phase reactions, where interiorsurfaces of through-holes are first modified with a vinylsilane film andfurther functionalized to attach a polyethylene glycol (PEG) film. Thekey characteristics of this approach is that once the exterior surfaceis coated with a hydrophobic silane film by stamping, the film is notaffected by a series of chemical reactions downstream targeted formodifying interior surfaces. Differential coating via stamping wassimple and straightforward for bench-level practice.

Different Exterior Coating on Opposite Sides of the Through-Hole Platen

A preferred embodiment of the differentially coated through-hole platenis to make the exterior coating on one surface of the platenfunctionally different from the exterior coating on the opposite surfaceand, furthermore, both exterior coatings are functionally different fromthe coatings on the interior surfaces of the through-holes.

One example of this embodiment is an exterior coating to facilitateliquid transfer between two through-hole plates spatially registered toalign the through-holes and brought into contact with each other. Onesurface of the plate is coated with a pressure-sensitive adhesive whilstthe opposite face is coated to be hydrophobic. There are fiduciary markson the platen to aid the alignment of the through-holes between two ormore platens. The surface of at least two platens are aligned relativeto the fiduciary marks and brought into contact such that the adhesivesurface contacts the hydrophobic surface. If the through-holes of oneplaten are empty and the other filled with fluid, fluid is transferredfrom one platen to the other. Otherwise, fluid in the opposingthrough-holes mix when the platens are brought into contact. Curing ofthe adhesive results in the bonding of the two platens, forming aunitary structure. The application of this method would include additionof reagents in one platen to samples in a second platen. Bonding the twoplatens together simplifies subsequent processing steps like washing,incubation or addition of another platen containing another set ofreagents.

Description of Current OpenArray Device and System

The present system is developed for nanoliter PCR, called the OpenArray™system, and is based on a rectilinear array of 3072, thirty-threenanoliter through-holes in a stainless steel platen the size of astandard microscope slide (25 mm×75 mm). The through-holes are arrangedin a pattern of 48 sub-arrays on a pitch equal to the wells in a384-well microplate (4.5 mm) and with 64 channels per sub-array. Theplaten surface is chemically modified with a process to make the insidesurface of each channel hydrophilic and the outside surface hydrophobic.The differential hydrophilic-hydrophobic coating facilitates preciseloading (CV<2%) and isolated retention of fluid in every channel.

Workflow to implement a PCR assay follows multiple steps. Individualprimer pairs are transferred with an array of 48 slotted pinsmanipulated by a 4 axis robot from wells in a 384 well microplate toindividual channels in each of the 48 sub-arrays. This reformattingoperation takes place in an environmentally controlled chamber toprevent evaporative loss. Once a platen is fully populated with primerpairs, the solvent is evaporated in a controlled manner leaving theprimers immobilized in a matrix on the inside surface of eachthrough-hole.

Next, forty-eight previously prepared samples (DNA or cDNA) are mixedwith Taq polymerase mastermix (Roche LightCycler) and loaded into eachsub-array (one sample per sub-array) with a 48 pipette tip-baseddispensing device called the Array-in-Array. Once the sample andmastermix are loaded into each subarray, a plug of UV curable epoxyseals the array in a glass-walled case with an immiscible fluid thatprevents evaporation during thermal cycling. Three encased arrays areplaced on the flat block of an imaging thermal cycler (NT Cycler)programmed to thermally cycle and image the arrays according to aprogrammed real time PCR protocol. For SYBR Green RT-PCR, a fluorescenceimage of the three arrays is acquired during the extension phase of thethermal cycle protocol. In this example, the NT Cycler is capable ofperforming 9,216 real-time PCR analyses, including temperature meltcurves, in under three hours. For SNP genotyping with the Taqman SNPassay, multiple arrays are thermally cycled on a flat block thermalcycler and then fluorescently imaged in the NT Cycler as an endpointmeasurement to determine genotype.

Software for implementing real-time PCR controls the NT Cycler, performsimage analysis to extract from the image sequence SYBR Green fluorescentintensities from each through-hole at each thermal cycle and tabulatesthe data in a flat file for further analysis with software toolsprovided in the NT Cycler environment or by a third party. Asample-centric approach organizes data and displays the results of theC_(T) calculation, copy number and melt temperature estimate for eachprimer pair analyzed with the system. The genotyping application uses asimilar software workflow but displays correlation plots of thefluorescent signals, provides the workflow for semi-automated genotypingcalling and exports the data in a flat file to a database.

Other Coatings

Liquid phase vinyl deposition with 7—octenyltrichorosilane or10—undecenyltrichrorosilane was used to coat steel substrates. The useof vinyl silanes derivatives will replace stamping as the coatingmethod. The vinyl terminated layer yields a hydrophobic surface with acontact angle in the range from 90 to 105 degrees. The vinyl layer canbe chemically modified into more hydrophilic groups providing thefunctionalities for internal coatings.

Methods for PEGylating the Internal Channels

Several PEG-silanes derivatives were tested for their ability to protectthe internal surface in a chip. Some examples are methoxy-PEG silane MW2000, methoxy-PEG silane MW 500 and methoxy-PEG silane MW 10,000.

Steel Coating Using Liquid Phase Aldehyde Deposition

Triethoxysilylbutyraldehyde was used as an alternative coating method.By using an aldehyde terminated surface, PEGylation can be done in asingle step. The vinyl terminated surface needs to be oxidized prior toPEGylation, which can potentially damage the external surface.

Introduction of a Longer PEG Molecule to Protect the Channels

Methoxy-PEG-amine MW 5000 was used to replace a silane-PEG 2000 coating.The acid terminated surface obtained after vinyl oxidation was modifiedwith amine PEG using NHS/EDC chemistry.

External Surface Chemical Modification

The use of vinyl-silane deposition to modify the internal and externalsurface of a chip in a batch process produces a hydrophobic surface thatmay not pass stringent Q.C. tests in the imitation PCR buffer or WISKtest. To overcome this problem several post-modification methods havebeen tested. Some examples are acylation of the vinyl surface with anacyl halide and a Lewis acid catalyst in what is essentially aFriedel-Crafts reaction. In this case, palmitoyl chloride can be used inthe presence of aluminum chloride.

Thick Polymeric Fillings

To improve the internal surface coatings, several methods have beentested. Crosslinking PEG inside the channels using hyperbranched PEG andPEI molecules and appropriate crosslinker molecules yielded greathydrophilic channels with excellent loading.

Aldehyde—Based Batch Process

This process represents an alternative to the current vinyl batchprocess to remove some potential coating problems during oxidation.PEGylation of the aldehyde activated surface using a Schiff's basemechanism yields better surface coating than EDC/NHS chemistry asapplied in the oxidized vinyl chemistry.

In Situ Polymerization Using a Vinyl Terminated Surface

This process employs the use of Polyethylene glycol 400, monoethylethermonomethacrylate (PEGMA) to PEGylate the surface by redox initiation inthe presence of AIBN.

Reversed Batch Process

This process inverts the steps in the process to minimize the effect ofoxidation on the external surface. Instead of vinyl, oxidation,PEGylation and perfluoro deposition, in that order, the vinyl surface isprotected first to run secondary deposition, followed by oxidation andPEGylation of the internal channels.

Surface Initiated PEG Attachment

Thiol PEG can be reacted with the vinyl terminated surface using atomtransfer by a radical-based reaction in the presence of copper bromideand bipyridine as catalyst.

Novel Coatings and Methods of Forming Coatings on Substrate IncludingStainless Steel

Formation of Cross-Linked Polyethylene Glycol Films

In this process, a surface exposes a functional group A. The surface isexposed to a solution containing a polyethylene glycol, whose end isterminated with a functional group B, which is reactive with afunctional group A. The solution also contains a polyethylene glycol,whose end is terminated with a functional group A. In addition, thesolution may contain a reagent that activates and/or assists theformation of covalent bonds between group A and B.

Example 1

A surface exposes a carboxylic acid moiety. A solution is prepared with6-arm polyethylene glycol terminated with —NH₂ and —COOH each and(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). Thecarboxylic acid-containing surface is exposed to the solution ofpolyethylene glycols. If necessary, the solution can be dried on thesurface and incubated at elevated temperature to improve the yield ofcross-linking between amine and carboxylic acid groups. The resultingsurface should show a thick, covalently-attached PEG film.

Formation of Thick Films of Polyelectrolytes by Layer-to-LayerAdsorption

A surface is exposed to a solution of polycations and polyanionssequentially. By electrostatic attraction, a film ofpolycation/polyanion is formed on the surface.

Differential Coating from Hydroxyl-Terminated Surface

The exterior and interior surfaces of OpenArray chip are uniformlycoated with a film exposing hydroxyl groups. Then, the chip is treatedwith standard batch coating process described elsewhere in theapplication.

Differential Coating from Epoxide-Terminated Surface

The surfaces of an OpenArray chip are uniformly coated with a filmexposing epoxide groups. Then, the surface is reacted with ammonia gasto open the ring and expose both hydroxyl and amine groups. A solutionof polyethylene glycol bearing amine-reactive functional groups isforce-loaded into through-holes only and incubated. Then, the chip istreated with standard batch coating process for differential coating, asdescribed elsewhere in the application.

Differential Coating from Amine-Terminated Surface

The surfaces of an OpenArray chip are uniformly coated with a filmexposing amine groups. A solution of polyethylene glycol bearingamine-reactive groups is forced-loaded into through-holes for selectivemodification. Then, the chip is processed with standard batch coatingprocess for differential coating, as described elsewhere in theapplication.

Formation of Cross-Linked Polyethylene Glycol Film Covalently Attachedto a Surface

A surface is modified with a reactive functional group, for example anepoxide group. A solution of polyethylene glycol bearing more than tworeactive functional groups, for example epoxide groups, is brought intocontact with the surface. Upon incubation, these reactive groups reactto form covalent bonds to each other to result in a covalently attachedand cross-linked polyethylene glycol film.

Formation of Polyethylene Glycol Film by Dendrimer Approach

A surface exposes a functional group X. A solution of PEG bearingreactive group Y is brought into contact with the surface, where the PEGreacts with the surface and still exposes many unreacted functionalgroups Y. Then, the surface is exposed to a solution of PEG bearinganother reactive group Z (or reactive group X) for covalent bondformation and immobilization of PEG. By repeating this step, acovalently bonded PEG film can be produced.

Formation of PEG Film by Polymer Brush Approach

A surface is functionalized with a group X and reacts with polymerchains by a grafting-to method. Then, a layer of PEG chains is attachedto form a polymer brush layer of PEG.

Photochemical Differential Surface Coating

Another method for creating differential surface coatings is to usephotochemistry to selectively modify the interiors of the channels (theetched or through-hole surfaces) relative to the array surfaces (theplanar non-etched or non-holed surfaces). To accomplish this, one musthave a method for selectively accessing different regions with light anda photochemically modified layer of molecules. The most practical methodfor optical access is the use of a photomask. The steel chips typicallyhave a narrow throat in the center of the through holes (a hourglassshape in cross section) thus increasing the ability for light to strikethe interior surfaces.

Example 2

The chip is coated with a hydrophobic silane and a photomask is placedover the chip such that light may only access the interior of thechannels. This assembly is exposed to a high energy UV light. Theassembly may be subjected to a flow of oxygen to create reactive oxygenspecies. The combination of light and reactive oxygen cleans the exposedsurfaces rendering them hydrophilic and capable of being furtherderivatized.

Example 3

The chip is coated with a hydrophilic silane such as a PEG silane and aset of photomasks is placed over the chip such that light may onlyaccess the exterior surfaces of the array. The array is then exposed toa hydrophobic silane to create the patterned array.

In such a process, patterned substrates of metal, particularly stainlesssteel chips, are created through the process of photochemically etchingfrom both sides. This two-sided etching of the through-hole wallscreates an hourglass shape for the through-holes, —when viewed incross-section. The resulting “throat” created in the metal chip may beemployed for advantage in that one may differentially react, or treat,parts of the channel interior. For example, the top half of the channelcould be left hydrophobic, and the bottom half cleaned tobecome—hydrophilic, then optionally coated with a PEG silane.Alternately, a silane with a photoactivated linker moiety may beemployed to create reactivity toward a modifying reagent. This methodcould be used to put capture probes such as oligonucleotides onto thearray and then to place encapsulated reagents in the array such thatthey do not block binding of analyte to the probes.

Alternative Force Filling Ideas

In addition to use of ethanol to accomplish force-filling of the etchedor through-hole surfaces, it is possibly to tightly stackhydrophobically coated chips and force reactive liquid into the etchedor through-hole surfaces. In such a method, the top and bottom chip of astacked array of chips will be sacrificed, but the rest will end up withthe desired reagents successfully applied in the etched or through-holesurfaces.

Alternatively, instead of force filling with modifying chemicals, theetched or through-hole surfaces can be force filled with etchingchemicals such as 1M potassium hydroxide or 1M sulfuric acid.

Example 4—Coating Autoloader

The coating autoloader is a method and device that enables fillingthrough-holes or microwells contained within a platen with a surfacemodifying or coating agent or agents (the reactive fluid), and thenprevents the reactive fluid from evaporating while the surfacemodification or coating agent/s perform their desired action. Thismethod and device results in highly uniform loading of the reactivefluid into the through-holes or microwells while minimizing the totalvolume of reagent is required.

In this embodiment, a chamber is provided to contain reagent fluid andplatens. In a preferred form the chamber is large enough the completelycontain eight through-hole plates. For example, chambers we havesuccessfully designed and used a chamber capable of coating 8 sheets×12arrays/sheet to give 96 open arrays. The chamber provides grooves orother mounts to hold the plurality of plates within the chamber, suchthat the plates are oriented in a substantially vertical direction andsuch that sufficient space is provided between plate faces for liquid toflow between them and between the plate faces and the chamber walls. SeeFIGS. 24A and 24 B for illustrations of such a device.

At the same time, minimizing the width of the chamber within theselimits reduces the volume of reactive fluid needed. The two large facesof the chamber are preferably made from a clear material such as glass,or polystyrene to permit observation of filling process. If necessarythe chamber may be held in a vertical orientation by clamps or a stand.The bottom of the chamber has one or more inlet ports through which thetransport fluid enters or exits.

A sufficient volume of surface modifying or coating agent or agents(reactive fluids) are introduced into the bottom of the chamber to forma thin layer. Next, an inert immiscible liquid or higher density liquid,such as mineral oil or a perfluorinated fluid, is introduced through theinlet port(s) into the bottom of the chamber below the first layer. Asthis ‘transport’ fluid is pumped into the bottom of the chamber, therising level lifts the surface modifying or coating agent or agentslayer so that it passes plates in a continuous manner. The flow oftransport fluid into the chamber must be smooth and controlled in speed;a peristaltic pump connected to a baffle to reduce turbulence is asuitable means for satisfying these requirements.

The volume of reactive fluid added to the chamber must be sufficientsuch that after volume needed to fill all desired through-holes has beenremoved, enough sample fluid remains in the chamber to form a continuouslayer that spans the chamber. This is done so that a continuous layer ofreactive fluid is maintained at all times during the loading process. Asdescribed elsewhere, the reactive fluid will enter the microwells orthrough-holes either though capillary action, or by exchange with alow-surface energy fluid that has been placed into all such volumesthrough various force-loading techniques. As the transport fluid levelrises in the chamber additional through-holes are filled with reactivefluid and then are subsequently submerged in the immiscible transportfluid.

Once the desired through-holes have been filled with reactive fluid, theexcess reagents can be drawn off the surface of the transport fluid atthe top of the chamber by means of suction, gravity, or capillaryaction. The transport fluid remaining in the chamber provides anenvironmental barrier between the loaded volumes of reactive fluid andthe surrounding atmosphere. Thus the plates may be incubated in thechamber while the desired physiochemical modification of the surfacetakes place without danger of evaporation. If a transport fluidappropriate vapor pressure and or melting point is used, the chamber mayeven be heated or cooled to control the reaction kinetics. Afterincubation, the ‘transport’ fluid is released from the chamber, out ofthe port(s) in the bottom.

Advantages of this device and method are this. (1) The process anddevice produces highly reproducible and uniform loading of the reactivefluid into the through-holes. This results in uniform surface propertiesinside the through-holes. (2) The volume of reactive fluid required isminimized producing cost savings on reagents. Although much largervolumes of transport fluid is required, the properties of immiscibilityand inertness allow this liquid to be reused and recycled. (3) Duringthe loading process, the contact time between the plate exteriorsurfaces and the reactive agent(s) is minimized; whereas after theloading is completed the contact time between the through-hole interiorsand the reactive agents can be maximized by incubating the plates in thetransport fluid. And (4), the process and device are scaleable andsuitable for automation.

Other methods for coating the external or internal surfaces of theplaten include sputtering gold onto all the surfaces of the patternedsubstrate followed by derivatization with hydrophobic thiols, afterwhich one can selectively react the derivatized etched or through-holesurfaces with hydroxyl reactive species.

In addition, variations employing a combination of the above-describedprocesses for preparing differential coatings on patterned substratesurfaces are envisioned that fall within the scope and spirit of thepresently described invention.

Thick Polymeric Fillings Multiple Layers

To improve the physic-chemical properties of the internal coatings,several methods have been tested. For instance, casting multiple polymerlayers inside the channels using hyper-branched PEG derivatives or PEIfor example and appropriate cross-linkers yielded a thick, multiplelayer polymeric coating.

Example 5—Coating Methods for Forming Multiple Layers Example5A—Formation of Multiple Polyethylene Glycol Films

A surface exposes a functional group X. A solution of a branched PEGderivative bearing reactive group Y is brought into contact with thesurface, where the PEG reacts with the surface and still exposes manyunreacted functional groups Y. In addition, the solution could contain areagent that activates and/or assists the formation of covalent bondsbetween group X and Y. Then, the surface is exposed to a second solutionof a branched PEG derivative bearing another reactive group Z (orreactive group X) for covalent bond formation and immobilization of PEG.By repeating this step, a covalently bonded multiple PEG films can beproduced. The process could be repeated several times until the desiredphysico-chemical properties are achieved.

Example 5B

A surface exposes a carboxylic acid moiety. A solution is prepared with6-arm amine terminated polyethylene glycol and EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride). Thecarboxylic acid-containing surface is exposed to the solution ofpolyethylene glycol. If necessary, the solution can be dried on thesurface and incubated at elevated temperature to improve the yield ofreaction between amine and carboxylic acid groups. The resulting surfaceshould show a thick, covalently-attached PEG film. The remaining aminegroups could be reacted, with a second solution prepared with 6-armpolyethylene glycol terminated with carboxylic groups and EDC togenerate a second polymeric layer. The process could be repeated severaltimes until the desired physico-chemical properties are achieved.

Example 5C—Formation of Cross-Linked Polyethylene Glycol Film CovalentlyAttached to a Surface

A surface is modified with a reactive functional group, for example anepoxide group. A solution of polyethylene glycol bearing more than tworeactive functional groups, for example epoxide groups, is brought intocontact with the surface. Upon incubation, these reactive groups reactto form covalent bonds to each other to result in a covalently attachedand cross-linked polyethylene glycol film.

Example 6 (FIGS. 24A-24C)

The stainless steel chip is supplied by a manufacturer in a format of 12chips in 6″×6″ sheet and processed as such throughout the whole coatingand reformatting (addition of reagents from microplates) proceduresuntil an individual chip is cut out for packaging. It is made of 316Lstainless steel and through-holes are formed via photographic patterningand selective two-sided simultaneous etching of exposed area by chemicaletchants. Upon arrival at BioTrove, sheets are checked for flatness,surface finish and array/sheet geometry using a combination of visualinspection and automated optical metrology. A sheet that does not meetspecs, even if only a single chip contains a defective spot, will beidentified and rejected. Whenever need arises, i.e. sheets manufacturedby new process or with new stock material, sample of sheets are sent forXPS analysis to confirm elemental composition of steel surfaces.Inspection results are entered into database.

Labeling

Currently, each sheet and chip is labeled manually for tracking andrecord keeping. A laser bar-coder and scanner will soon be implementedfor all chips to automate a tracking system. A laser bar-coder inscribesbar codes and alphanumerics on each chip and scanners read the codes andrecord the information at each major step in chip manufacturing. Thissystem facilitates access to process and handling information for anindividual sheet and chip, as well as provides a database link to thecustomer for the contents of each well in the chip.

Cleaning

Sheets are cleaned by exposure to 10% RBS® aqueous solution at 50° C.for two hours with agitation. RBS® is a basic detergent, available fromBacto Laboratories Pty LTD, containing non-ionic and ionic surfactantsand widely used in cleaning glass substrates. Cleaned sheets are rinsedin NaCl salt solution and deionized (DI) water bath sequentiallyfollowed by a cascade rinsing in counterflow. This method of cascaderinsing consists of a series of rinse tanks which are plumbed to causewater to flow from one tank to another in the direction opposite to thatof the workflow. After cascade rinsing, the sheets are briefly immersedin a hot ethanol bath and placed in a preheated vacuum oven at 50° C.for drying under a stream of N₂. Alternatively, drying can be donemanually by using a N₂ gun.

Coating of Steel Sheets with Vinyl-Terminated Silane

Surfaces of steel sheets are coated uniformly with vinyl-terminatedsilane by vapor-phase deposition. Cleaned sheets are placed in a customoven along with appropriate amount of 7-octenyltrimethoxysilane in anopen container. The oven is operated under vacuum after previouslypurging with nitrogen gas twice and heated to 100° C. for 5 h whilecompletely isolated. 7-octenyltrimethoxysilane is then completelyevaporated from its container and forms a molecular-level film onsurface. Once thermal deposition of the silane on the surface is done,the chamber is purged with nitrogen gas and placed under vacuum again toremove the evaporated silane. Then, an appropriate amount of ammonia gasis introduced into the chamber in order to cure the silane film on thesurfaces and enhance the stability and robustness of the film. Sheetscoated with the vinyl-terminated silane are sealed in a vacuum packagedaluminized Mylar pouch for storage until the next step.

The presence of the 7-octenyltrimethoxysilane film is confirmed by highcontact angle of water, ˜100° C., of a modified sheet and newSilicon(2s) and Silicon(2p) signals and increased Carbon(1s) signal inX-ray Photoelectron Spectroscopy (XPS) analysis. Further, when a siliconslide was treated under an identical condition side-by-side with steelsheets, the slide exhibited a film of −1 nm thickness by ellipsometry,corresponding to monolayer coverage of the silane. It is concludedtherefore that the vapor-phase deposition of 7-octenyltrimethoxysilaneproduces a monolayer-level of the silane film on steel surfaces.

In manufacturing setting, silane-based surface modification byvapor-phase deposition has many advantages over solution-phase method.It is well known that surface modification employing silane reagents isextremely sensitive toward environmental conditions and tricky toreproduce in the long run, particularly in bulk scale. Between twomethods of silane surface modification, vapor-phase method is moretolerant to environmental conditions and easier to control criticalreaction parameters and, therefore, superior in achieving betteruniformity, consistency, and reproducibility. In addition, because thevapor-phase method does not need any solvent for silane reaction andpost-modification rinsing, it is much more cost-effective andenvironmentally safe.

Selective Oxidation of Through-Holes Surfaces

Selective oxidation of the internal vinyl-terminated surface is achievedby force loading a permanganate solution into the channels. Forceloading is required because of the hydrophobic nature of the vinylsurface. The method of force loading used here is to immerse the sheetof arrays in a bath of low surface energy liquid such as ethanol. Theliquid will flood all of the surfaces of the array including thethrough-holes. When the sheet is withdrawn vertically from the bath,ethanol remains in the through-holes yet drips off of the surfaces. Thearray is then immersed into an aqueous solution to replace the ethanolwith aqueous solution. When withdrawn, the array holds the polar(aqueous) solvent in the holes despite being initially hydrophobic onboth interior and exterior array surfaces. Other methods of forcefilling include filling under vacuum, under hydrostatic pressure, orunder a flowing liquid. For the permanganate oxidation, the conditionsof time, oxidant concentration and temperature must be carefully chosento prevent extensive oxidation of the exterior array surfaces.

In practice, a fresh oxidation solution consisting of 5 mM KMnO₄ and19.5 mM NaIO₄ in reverse osmosis deionized (RODI) water is used tooxidize surface inside the channels. To force load the oxidant into theholes, a sheet is submerged in ethanol for about 30 seconds, followed byRODI water, and oxidation solution. The sheet with the holes loaded withoxidant is then incubated for 2 hours under perfluorinated liquid Afteroxidation, the sheet is rinsed with 0.3 M NaHSO₃ to reduce any excesspermanganate and followed by an acid rinse to protonate and protect theacid terminated surface.

Selective PEGylation of Through-Hole Surfaces

PEGylation of the acid terminated layer is performed by reactingamine-terminated PEG MW 5000 in the presence of(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide.HCl (EDC) as a catalyst.After an incubation period of 2 hours at room temperature underFluorinert, the PEG in the channels is allowed to dry overnight at 100°C. By doing this, the PEGylation efficiency is dramatically improved asobserved by XPS analysis.

Reloading of PEG onto Through-Holes

Prior to secondary deposition, the PEGylated surface is rinsed in RODIwater to remove any EDC/PEG residues. However, to protect the channelsfrom perfluorosilane deposition, a PEG reload was found to be necessary.A fresh 50 mg/mL1 PEG 8000 is used to reload and protect the channel.After loading the sheets are placed in an oven at 50° C. under vacuumfor one hour to dry a protective PEG layer on the inside of the holes.

Coating of Steel Sheets with Perfluorosilane Selectively Outside ofThrough-Holes

A film of heptadecafluoro-1,1,2,2-tetrahydrodecyltriethoxysilane (a.k.a.perfluorosilane) is formed uniformly on exteriors and interiors ofthrough-holes by vapor-phase deposition. The deposition is performed ina similar manner as vinyl-terminated silane deposition described above.Sheets are placed in a chamber of a vacuum oven along with appropriateamount of perfluorosilane in an open container. Dedicated ovens are usedfor the two different vapor phase steps. After putting the chamber undervacuum, it is heated to 150° C. for 2 hours followed by ammonia curingstep to secure the deposited film on a surface. The coated sheets arerinsed in NaCl solution and a series of DI water bath sequentially toremove PEG dried in through-holes from “Reloading of PEG” step.Naturally, rinsing away dried PEG in through-holes removesperfluorosilane film deposited on its top and ends up exposing a freshcovalently attached PEG layer. In contrast, perfluorosilane filmdeposited on exterior surfaces remains through extensive rinsing stepsand exhibits the hydrophobicity and oleophobicity of a typicalperfluorosilane film. Finished sheets bear hydrophilic, biologicallyinert PEG film on through-holes surfaces and hydrophobic perfluorosilanefilm on exterior surfaces.

A perfluorosilane film formed by vapor-phase method was examined bycontact angle measurements, XPS, and ellipsometry. In XPS spectra, thefilm on both steel and silicon surfaces showed characteristic pattern ofpeaks expected from a typical perfluorosilane monolayer. Theellipsometrical thickness of the silane film on silicon slide preparedunder identical condition side-by-side with steel sheets was ˜1.2 nm.The contact angle of water on perfluorosilane films formed on top ofvinyl-terminated film or bare surface was higher than 110°. Based on allthe data collected, it is confirmed that vapor-phase deposition ofperfluorosilane produces a molecular film of monolayer coverage.

What is claimed is:
 1. A method for differentially coating a substrate,the method comprising: supplying a substrate comprising an externalsurface and a plurality of through-hole channels each comprising aninterior surface in communication with an exterior surface; applying afirst coating agent simultaneously to both the exterior surface and toat least one of the interior surfaces; selectively oxidizing the atleast one interior surface by loading the at least one interior surfacewith an aqueous solvent; after selectively oxidizing, selectivelymodifying the first coating agent applied to the at least one interiorsurface with a first modifying agent such that, after modifying, the atleast one interior surface has a first specified physicochemicalproperty that differs with respect to a specified physicochemicalproperty of the external surface; and applying a second modifying agentto the at least one interior surface; wherein: the aqueous solventcomprises ethanol; the aqueous solvent comprises permanganate solution;the aqueous solvent comprises a cross-linking agent; or the methodfurther comprises, after selectively oxidizing, incubating the substratein a nonreactive oil or liquid.
 2. The method of claim 1, wherein atleast one of (1) the first coating agent comprises a vinyl terminatedcompound or (2) the first and second modifying agents each comprise apolyethylene glycol.
 3. The method of claim 1, wherein the aqueoussolvent comprises permanganate solution KMnO₄ and NaIO₄ in deionizedwater.
 4. The method of claim 1, wherein the aqueous solvent solutioncomprises permanganate solution 5 mM KMnO₄ and 19.5 mM NaIO₄ indeionized water.
 5. The method of claim 1, wherein the nonreactive oilor liquid is a perfluorinated alkane solution.
 6. A method fordifferentially coating a substrate, the method comprising: supplying asubstrate comprising an external surface and a plurality of through-holechannels each comprising an interior surface in communication with anexterior surface; applying a first coating agent simultaneously to boththe exterior surface and to at least one of the interior surfaces;selectively oxidizing the at least one interior surface by loading theat least one interior surface with a liquid modifying agent; afterselectively oxidizing, selectively modifying the first coating agentapplied to the at least one interior surface with a first modifyingagent such that, after modifying, the at least one interior surface hasa first specified physicochemical property that differs with respect toa specified physicochemical property of the external surface; andapplying a second modifying agent to the at least one interior surface;wherein: the liquid modifying agent comprises ethanol; the liquidmodifying agent comprises permanganate solution; the liquid modifyingagent comprises a cross-linking agent; or the method further comprises,after selectively oxidizing, incubating the substrate in a nonreactiveoil or liquid.
 7. The method of claim 6, wherein at least one of (1) thefirst coating agent comprises a vinyl terminated compound or (2) thefirst and second modifying agents each comprise a polyethylene glycol.8. The method of claim 6, wherein the liquid modifying agent comprisespermanganate solution KMnO₄ and NaIO₄ in deionized water.
 9. The methodof claim 6, wherein the liquid modifying agent comprises permanganatesolution 5 mM KMnO₄ and 19.5 mM NaIO₄ in deionized water.
 10. The methodof claim 6, wherein the nonreactive oil or liquid is a perfluorinatedalkane solution.